Imatinib is an effective first-line therapy for chronic myelogenous leukemia (CML) that acts by targeting the tyrosine kinase activity of BCR-ABL. To overcome resistance, second-generation inhibitors of BCR-ABL have been developed. Among these, nilotinib is more potent against BCR-ABL than imatinib, and is effective against many imatinib-resistant BCR-ABL mutants. In this study, an in vitro flow cytometry assay to analyze imatinib- and nilotinib-induced apoptosis in CML cells has been developed. Both the drugs induced significant apoptosis in CD34+ cells from 36 CML bone marrow samples (P<10(-4)), whereas CD34+ cells from BCR-ABL negative samples were unaffected. When the experiments were carried out in the presence of a cocktail of cytokines, nilotinib- but not imatinib-induced apoptosis was inhibited. This differential inhibition was confirmed on K562 cells. A blocking anti-CD117 antibody alleviated the antiapoptotic effect of cytokines against nilotinib. Moreover, using short hairpin RNA against BCR-ABL, we showed that K562 cells were not dependent on BCR-ABL signaling as long as the stem cell factor (SCF) receptor pathway was activated. We conclude that the c-KIT pathway may substitute for BCR-ABL tyrosine kinase to activate survival signals, and that c-KIT must be inhibited besides Bcr-Abl to allow apoptosis of CML cells.