Two distinct CD95 (APO-1/Fas) signaling pathways have been identified in cell types that use either pathway almost exclusively. Type I cells activate caspase-8 within seconds and caspase-3 within 30 minutes of CD95 engagement, while type II cells delay caspase activation for ~60 minutes. Both cell types show similar kinetics of CD95-mediated apoptosis and mitochondrial transmembrane potential (ΔΨm) loss. Overexpression of Bcl-2 or Bcl-xL blocks mitochondrial apoptotic activities in both cell types, but only in type II cells does it block caspase-8 and caspase-3 activation and apoptosis. In type I cells, caspase-8 is activated by the death-inducing signaling complex (DISC), leading to caspase-3 activation. In type II cells, DISC formation is reduced, and caspase-8 and caspase-3 activation occurs after ΔΨm loss. Overexpression of caspase-3 in the caspase-3-negative cell line MCF7-Fas converts these cells into true type I cells, where apoptosis is no longer inhibited by Bcl-xL. The amount of active caspase-8 generated at the DISC determines whether a mitochondria-independent apoptosis pathway is used (type I cells) or not (type II cells). Mitochondria are similarly activated in both cell types during CD95-mediated apoptosis, but type II cells depend on mitochondrial contribution to apoptosis. Bcl-2 overexpression blocks CD95-mediated apoptosis in type II cells but not in type I cells. Caspase-8 activation in type I cells occurs upstream of mitochondria, while in type II cells it occurs downstream. DISC formation is strongly reduced in type II cells, and caspase-8 and caspase-3 activation occurs after ΔΨm loss. The presence of caspase-3 enables type II cells to bypass mitochondrial functions. The two CD95 signaling pathways are evolutionarily distinct, with type II cells resembling the C. elegans apoptosis pathway, while type I cells use a mitochondria-independent pathway. The physiological roles of these pathways include type I cells in peripheral T cells and thymocytes, and type II cells in tissues like the liver. Bcl-2 and Bcl-xL overexpression inhibit CD95-mediated apoptosis in type II cells but not in type I cells. The amount of active caspase-8 generated at the DISC determines whether a cell uses a mitochondria-independent or -dependent apoptosis pathway.Two distinct CD95 (APO-1/Fas) signaling pathways have been identified in cell types that use either pathway almost exclusively. Type I cells activate caspase-8 within seconds and caspase-3 within 30 minutes of CD95 engagement, while type II cells delay caspase activation for ~60 minutes. Both cell types show similar kinetics of CD95-mediated apoptosis and mitochondrial transmembrane potential (ΔΨm) loss. Overexpression of Bcl-2 or Bcl-xL blocks mitochondrial apoptotic activities in both cell types, but only in type II cells does it block caspase-8 and caspase-3 activation and apoptosis. In type I cells, caspase-8 is activated by the death-inducing signaling complex (DISC), leading to caspase-3 activation. In type II cells, DISC formation is reduced, and caspase-8 and caspase-3 activation occurs after ΔΨm loss. Overexpression of caspase-3 in the caspase-3-negative cell line MCF7-Fas converts these cells into true type I cells, where apoptosis is no longer inhibited by Bcl-xL. The amount of active caspase-8 generated at the DISC determines whether a mitochondria-independent apoptosis pathway is used (type I cells) or not (type II cells). Mitochondria are similarly activated in both cell types during CD95-mediated apoptosis, but type II cells depend on mitochondrial contribution to apoptosis. Bcl-2 overexpression blocks CD95-mediated apoptosis in type II cells but not in type I cells. Caspase-8 activation in type I cells occurs upstream of mitochondria, while in type II cells it occurs downstream. DISC formation is strongly reduced in type II cells, and caspase-8 and caspase-3 activation occurs after ΔΨm loss. The presence of caspase-3 enables type II cells to bypass mitochondrial functions. The two CD95 signaling pathways are evolutionarily distinct, with type II cells resembling the C. elegans apoptosis pathway, while type I cells use a mitochondria-independent pathway. The physiological roles of these pathways include type I cells in peripheral T cells and thymocytes, and type II cells in tissues like the liver. Bcl-2 and Bcl-xL overexpression inhibit CD95-mediated apoptosis in type II cells but not in type I cells. The amount of active caspase-8 generated at the DISC determines whether a cell uses a mitochondria-independent or -dependent apoptosis pathway.