Interleukin-3 (IL-3) deprivation of the mouse pro-B cell line Ba/F3 induces cell death that is abrogated by B-cell lymphoma 2 (Bcl-2) overexpression, but remains unaffected by the pan-caspase inhibitor carbobenzoxy-valyl-analyl-aspartyl-[O-methyl]-fluoromethylketone (zVAD-fmk). IL-3 withdrawal causes receptor-interacting protein (RIP)1 cleavage into C-terminal fragments of 30 and 25 kDa, and only cleavage leading to the former was prevented by zVAD-fmk. siRNA experiments demonstrated that generation of the 25-kDa fragment was due to a Bcl-2-modulated release of the mitochondrial serine protease high temperature requirement protein A2 (HtrA2)/Omi. Accordingly, recombinant HtrA2/Omi efficiently cleaved mouse RIP1 in vitro, generating fragments matching those observed in IL-3-deprived Ba/F3 cells. The HtrA2/Omi cleavage site in mouse RIP1 was mapped to the intermediate domain and the corresponding N- and C-terminal fragments were impaired in their ability to activate nuclear factor-kappaB, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase. Interestingly, knockdown of HtrA2/Omi afforded protection against IL-3 withdrawal-induced death in the presence of zVAD-fmk, demonstrating a role for HtrA2/Omi in caspase-independent cell death during growth factor withdrawal by cleaving RIP1.