Taxane derivatives such as paclitaxel elicit their antitumor effects at least in part by induction of apoptosis, but the underlying mechanisms are incompletely understood. Here, we used different cellular models with deficiencies in key regulators of apoptosis to elucidate the mechanism of paclitaxel-induced cell death. Apoptosis by paclitaxel was reported to depend on the activation of the initiator caspase-10; however, we clearly demonstrate that paclitaxel kills murine embryonic fibroblasts (MEFs) devoid of caspase-10 as well as human tumor cell lines deficient in caspase-10, caspase-8, or Fas-associating protein with death domain. In contrast, the lack of Apaf-1 or caspase-9, key regulators of the mitochondrial pathway, not only entirely protected against paclitaxel-induced apoptosis but could even confer clonogenic survival, depending on the cell type and drug concentration. Thus, paclitaxel triggers apoptosis not through caspase-10, but via caspase-9 activation at the apoptosome. This conclusion is supported by the fact that Bcl-2–overexpressing cells and Bax/Bak doubly-deficient MEFs were entirely resistant to paclitaxel-induced apoptosis. Interestingly, also the single knockout of Bim or Bax, but not that of Bak or Bid, conferred partial resistance, suggesting a particular role of these mediators in the cell-death pathway activated by paclitaxel.