Mitochondria play a crucial role in activating apoptosis in mammalian cells. Bcl-2 family members regulate the release of proteins from the intermembrane space between the mitochondrial inner and outer membranes, which then activate caspase proteases that dismantle cells and signal efficient phagocytosis of cell corpses. This review discusses the extensive literature on proteins released from the intermembrane space and considers genetic evidence for and against their roles in apoptosis activation. It also compares and contrasts apoptosis pathways in *Caenorhabditis elegans*, *Drosophila melanogaster*, and mammals, highlighting major mysteries that remain to be solved. Key mitochondrial proteins involved in apoptosis include cytochrome c (Cyt c), Smac, Omi, EndoG, and AIF. Cyt c is essential for caspase activation and is released from mitochondria during apoptosis. Smac and Omi are released to relieve the inhibitory effects of inhibitors of apoptosis (IAPs) on caspase activity. EndoG and AIF are also released from mitochondria and play roles in DNA/RNA degradation and chromatin condensation, respectively. The role of mitochondria in apoptosis is less clear in invertebrates, with limited evidence for their essential roles in *C. elegans* and *D. melanogaster*. The review highlights the need for further research to understand the evolutionary conservation and mechanisms of apoptosis in different organisms.Mitochondria play a crucial role in activating apoptosis in mammalian cells. Bcl-2 family members regulate the release of proteins from the intermembrane space between the mitochondrial inner and outer membranes, which then activate caspase proteases that dismantle cells and signal efficient phagocytosis of cell corpses. This review discusses the extensive literature on proteins released from the intermembrane space and considers genetic evidence for and against their roles in apoptosis activation. It also compares and contrasts apoptosis pathways in *Caenorhabditis elegans*, *Drosophila melanogaster*, and mammals, highlighting major mysteries that remain to be solved. Key mitochondrial proteins involved in apoptosis include cytochrome c (Cyt c), Smac, Omi, EndoG, and AIF. Cyt c is essential for caspase activation and is released from mitochondria during apoptosis. Smac and Omi are released to relieve the inhibitory effects of inhibitors of apoptosis (IAPs) on caspase activity. EndoG and AIF are also released from mitochondria and play roles in DNA/RNA degradation and chromatin condensation, respectively. The role of mitochondria in apoptosis is less clear in invertebrates, with limited evidence for their essential roles in *C. elegans* and *D. melanogaster*. The review highlights the need for further research to understand the evolutionary conservation and mechanisms of apoptosis in different organisms.