BAX and BAK are “multidomain” proapoptotic proteins that initiate mitochondrial dysfunction but also localize to the endoplasmic reticulum (ER). Mouse embryonic fibroblasts deficient for BAX and BAK (DKO cells) were found to have a reduced resting concentration of calcium in the ER ([Ca²⁺]_er) that results in decreased uptake of Ca²⁺ by mitochondria after Ca²⁺ release from the ER. Expression of SERCA (sarcoplasmic-endoplasmic reticulum Ca²⁺ adenosine triphosphatase) corrected [Ca²⁺]_er and mitochondrial Ca²⁺ uptake in DKO cells, restoring apoptotic death in response to agents that release Ca²⁺ from intracellular stores (such as arachidonic acid, C₂-ceramide, and oxidative stress). In contrast, targeting of BAX to mitochondria selectively restored apoptosis to “BH3-only” signals. A third set of stimuli, including many intrinsic signals, required both ER-released Ca²⁺ and the presence of mitochondrial BAX or BAK to fully restore apoptosis. Thus, BAX and BAK operate in both the ER and mitochondria as an essential gateway for selected apoptotic signals.