The Sacrifice Room is a special room added in the Wrath of the Lamb expansion. It is a room with the same backdrop as The Depths, but with numerous pools of blood scattered around. In the center of the room, there is a Spike tile. Touching it will deal a full heart of damage to Isaac, even if he has Flight. After touching it enough times, a Chest will spawn, which can be regular, Golden, or Red. A chest will always spawn if Isaac's health is reduced to a half-heart.
Only one chest will be rewarded per Sacrifice Room.
Using items like Book Of Shadows, My Little Unicorn, or The Gamekid when passing over the spikes will render the player invulnerable to the spikes and quickly spawn the chest, regardless of the player's current health (This has been removed in Rebirth). Using the brief invulnerability that occurs upon taking damage to walk over the spikes, such as through the use of the IV Bag, will not result in the chest spawning, nor will flying over the spikes during the temporary invulnerability gained from the spacebar functionality of A Pony.
??? is treated normally when using this (unlike other rooms/items that consider him always at half a heart).
Sacrifice rooms are more likely to be spawned if the player has full HP (Soul hearts will add up as well).
If the player has one half of a red heart and one soul heart remaining, stepping on the spikes does not guarantee a chest spawn, despite the spikes reducing health to one half of a heart.