Dean: Why break the seal anyway?
Castiel: You think of the seals as locks on a door.Castiel: Lucifer walks free.
Dean: Okay, last one opens, and?
Lucifer cannot rise. He does and Hell rises with him.
The 66 Seals are special attachments made by God to secure Lucifer's Cage. The seals are all represented by important events, the first of which was the shedding of blood by a righteous man in Hell and the last of which was Lilith's death. When these attachments broke, Lucifer's Cage was immediately opened and he was released. According to Anna, there were about 600 possible seals of which only 66 had to break for Lucifer to be freed. Due to Castiel's interactions with Dean and Sam, it is believed that Heaven is desperately trying to keep Lucifer contained and that the Angels are trying to stop Lilith from breaking the 66 Seals. Zachariah later reveals that he, along with the executive members in the Host, intended for all of the Seals following the first seal to break so that Lucifer could break free, hastening the epic battle between Michael and Lucifer.
- Castiel describes the seals as locks on a door that hold Lucifer prisoner. He also states that a group of demons, including Lilith, is trying to break them.
- Once Dean put the Apocalypse in motion by breaking the first seal, the main and executive Angels of Heaven intended for the rest of the seals to break.
- In addition to the 66 Seals, the rings belonging to the Four Horsemen of the Apocalypse can also open Lucifer's Cage.
- The First Seal: "The first seal shall be broken when a righteous man sheds blood in Hell." The demons intend for this to be John Winchester; instead it is Dean.
- Rise of the Witnesses.
- Raising of Samhain.
- Ten species going extinct in Key West.
- Everyone in a fifteen-man fishing crew going blind in Alaska.
- A teacher in New York killing sixty-six students.
- Death of two reapers on the night of the solstice. Must say "hic cruor messorius illud sigillum quod luciferem reverendum obstringit aperiat ut resurgat." Which translates to "This blood Reaper seal that binds to open reverend rise of Lucifer." (unbroken).
- The Final Seal: "The first demon will be the last seal"; Sam kills Lilith, Lucifer's first demon, which broke the final seal.
In this episode Lilith casts a spell to bring about the Rise of the Witnesses - people whom hunters failed to save. While Sam, Dean and Bobby stop the Witnesses, the seal is broken.
Castiel reveals that the 66 Seals are the key to unleashing the Apocalypse. Lilith is breaking them one by one. Even if the evil thing unleashed is quelled or defeated, the seal is not restored and Hell on Earth is one step closer.
The raising of Samhain breaks another seal.
It is revealed by Anna Milton that whilst 66 Seals need to be broken for Lilith's plan to succeed, there are around 600 possible seals. No one knows which ones she will attempt to break, making it nearly impossible for anyone, even the angels, to predict her actions or prevent them.
Ruby informs Sam that 34 of the 66 seals have been broken. She says the angels are losing, and urges him to kill Lilith.
Alastair is trying to break a seal that requires the death of two reapers on the night of the solstice. Castiel, pretending to be Bobby, tells Sam of this, informing Sam how it is described in a "version of Revelations":
- And he bloodied death under the newborn sky
- Sweet to taste
- But bitter once devoured 
Castiel needs Sam and Dean as the demons are holed up in a refuge protected by sigils that keep angels out. The boys are successful in stopping Alastair and saving the seal.
As Dean is torturing Alastair, Alastair reveals that Dean was responsible for the first seal breaking:
- Alastair: And it is written that the first seal shall be broken when a righteous man sheds blood in Hell. As he breaks, so shall it break. We had to break the first seal before any others, only way to get the dominoes to fall, right? Topple the one at the front of the line. When we win, when we bring on the Apocalypse and burn this earth down, we owe it all to you, Dean Winchester.
He also reveals that the demons hoped that John Winchester would be the man to break the first seal, but John didn't break under their torture and always refused to shed another's blood.
Castiel confirms that Dean is responsible for the first seal being broken, even though Heaven "laid siege" to Hell to try and rescue him. He tells Dean they raised him from Hell because only the righteous man who broke the first seal can "end it" - although he is unclear on the details.
Rufus Turner calls Bobby with news that other seals have been broken. The seals Rufus Turner reports being broken are: ten species going extinct in Key West, a fishing crew of fifteen going blind in Alaska, and a teacher in New York killing 66 students, all in one day. Bobby believes that there aren't many seals left and that maybe they should release Sam from his panic room to stop Lilith.
Ruby tells Sam there are only two or three seals left to be broken. She says only "Lucifer's first" can break the final seal - and that is Lilith. This renews Sam's desire to kill Lilith.
Lilith's death is the final seal, the one which will free Lucifer.
- Ruby: And as it is written, the first demon will be the last seal.
This fact is known to the higher angelic ranks, Lilith, and Ruby. Each in their own ways, they have all been manipulating Sam so that he will kill her. Castiel reveals the nature of the final seal to Dean and sends him to stop Sam, but Dean is unable to prevent Sam from killing Lilith. Lucifer is freed.
- Most of the Seals witnessed focus on a corruption of the natural order; a good man goes to Hell and tortures others, ghosts rise up to attack others when they were not naturally vengeful spirits, reapers - spirits who take the dead to the afterlife - are killed themselves, and a teacher kills the students he is responsible for.