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Challenges are special playthroughs where the player starts with a specific item or set up, and some limitation such as no Treasure Rooms or the Curse of Darkness for every floor. Upon completion of each challenge, the player unlocks a new item that can be found in the game, as well as unlocking a Steam achievement. Completing challenges also counts as beating the game, so a new ending will be unlocked upon completion.

Note: Challenges are first introduced in the Wrath of the Lamb expansion for the original game, and all original challenges are replaced by newer ones for Rebirth.

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