Most thymocytes that have not successfully rearranged their TCR genes or that express a receptor with subthreshold avidity for self-Ag/MHC enter a default apoptosis pathway, death by neglect. Spontaneous thymocyte apoptosis (STA), at least in part, may mimic this process in vitro. However, the molecular mechanism(s) by which thymocytes undergo this spontaneous apoptosis remains unknown. Here, we report that caspsase-1 and caspase-3 are activated during STA, but these caspases are dispensable for this apoptotic process. The inhibition of STA by a pan-caspase inhibitor, zVAD, suggests that multiple caspase pathways exist. Importantly, the early release of cytochrome c from mitochondria closely correlates with the degradation of Bcl-2 and Bcl-xL and a decrease in the ratios of Bcl-2 and Bcl-xL to Bax during STA. These findings suggest that the degradation of Bcl-2 and Bcl-xL may favor Bax to induce cytochrome c release from mitochondria, which subsequently activates downstream caspases in STA. Our data provide the first biochemical insight into the molecular mechanism of STA.