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The Gaper, Mulligen, Mulligoon, and The Swarmer are all the exact same enemies, but in different states of decay.
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On the character select screen, Cain's Lucky Foot item is misspelled as "Luck Foot."
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"I Am Error" is a rare, special room that only has a 6.66% chance of being accessible when using telepills. The name and appearance of the character in the room references Zelda II: The Adventure of Link.
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Some Mac users of the game have encountered a glitch where, on start up, the screen will remain black and the player will have maximum hearts, 99 cents, two trinkets named "Death", a book named "Death" in the consumable window, and the Bible icon in the item window.
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The item "Bible" can not only give you wings for usage of one room, but can also kill Mother during the fight with her, or you if you decide to use it while fighting Satan.
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To the left of every boss Health Bar (excluding Mask of Infamy) is an angry face. This is a reference to a character on the website Newgrounds called Mr. Angry Face.
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The posters in the arcade room are references to Edmund McMillen's previous games. Namely Meat Boy, Time Fcuk, Gish, Tri-Achnid, Spewer, Coil and Aether.
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There is a chance that the person found sitting in a secret room will have the face of the "Forever Alone" meme.
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The character "???" also known as the Blue Baby, is a reference to both Edmund McMillen's Newgrounds username, BlueBaby, and one of his earliest Newgrounds submissions, "Dead Baby Dressup".
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The fez on Judas' head is a referential jab at FEZ creator Phil Fish, who voted against Edmund McMillen and his team at the Independent Games Festival. The name "Judas" likely was intended to evoke Judas Iscariot of the New Testament, and suggests that the character Judas represents Fish's betrayal of Edmund.
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Completing the Chest with any character is supposed to unlock an item and an achievement after the first attempt. While the in-game award is correct, the Steam achievements are glitched, where beating the Chest with one character will unlock the achievement of another. This goes for all characters excluding Maggy and Isaac.
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Pausing the game immediately after triggering a fight in a challenge room will have all waves of enemies spawn at once. Florian Himsl will not fix this bug because he finds it funny.
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According to developer Edmund McMillen, the working title for The Binding of Isaac was "Love Sick" and was later changed to "Mom" before it eventually became The Binding of Isaac.
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As recently as the game's third update, the passive item "Money=Power" contained a typo that made it appear as "Monkey=Power". This would be patched out in a future update.
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The designs of the passive item Infamy and the boss Mask of Infamy are a reference to Carmilla from Castlevania II: Simon's Quest.
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