Histone H2AX plays an important role in regulation of tumor cell apoptosis and acts as a novel human tumor suppressor protein.However, it is still unknown how H2AX phosphorylation at Ser 139 and Tyr 142 is regulated in Chronic myelogenous leukemia (CML) cells.In this study, we uncovered the signaling transduction involved in H2AX phosphorylation and its requirement for apoptosis of CML cells.The results demonstrated that imatinib induced strong phosphorylation of H2AX at Ser139 and Tyr142 in a time-and dose-dependent manner in K562 cells.However, H2AX acetylation (Lys 5) was not affected by imatinib, although the acetylated H2AX maintained a higher endogenous level in K562 cells.Caspase-3 and its downstream mammalian STE20-like kinase 1 (Mst1) could be activated by imatinib during apoptosis, coinciding with H2AX phosphorylation at Ser139 and Tyr142.Inhibition of the caspase-3/Mst1 pathway with caspase-3 inhibitor Z-VAD reduced H2AX phosphorylation at Ser139 and Tyr 142 and blocked K562 cell apoptosis.In addition, we found that imatinib induced expression of Williams-Beuren syndrome transcription factor (WSTF) but not wild-type p53-induced phosphatase l(Wip 1).Taken together, these data show that the caspase3/Mst1 pathway is required for H2AX C-terminal phosphorylation (Ser139 and Tyr142) and subsequent apoptosis induced by imatinib in Bcr-Abl-positive K562 cells.