Bcr-Abl tyrosine kinase inhibitors (TKI) are effective in inducing remissions in chronic myelogenous leukemia (CML) patients but do not eliminate primitive CML hematopoietic cells. There is a need to identify mechanisms that contribute to retention of CML progenitors. Src family tyrosine kinases have been identified as potential mediators of Bcr-Abl–induced leukemogenesis. Dasatinib (BMS-354825) is a potent dual Abl/Src kinase inhibitor approved for clinical use in CML patients. We evaluated Src activity in primitive human CML progenitors from different stages of disease and investigated effects of Dasatinib on Src activity and downstream signaling pathways. P-Src expression was increased in CD34⁺ cells and CD34⁺CD38⁻ cells in all phases of CML. Dasatinib showed potent Src inhibitory activity in CML progenitors, inhibiting both Bcr-Abl–dependent and –independent Src activity. In contrast, Imatinib inhibited only Bcr-Abl–dependent Src activity. Dasatinib inhibited P–mitogen-activated protein kinase (MAPK), P-Akt, and P-STAT5 levels in CML progenitors in the absence of growth factors but not in the presence of growth factors. A marked increase in P-MAPK levels seen in the presence of growth factors with Imatinib was much less prominent with Dasatinib. Dasatinib significantly suppressed CML colony-forming cells and long-term culture-initiating cells but did not significantly alter the level of apoptosis-regulating proteins in CML CD34+ cells. Our results indicate that Dasatinib, in addition to potent anti–Bcr-Abl kinase activity, effectively inhibits Src kinase activity and downstream signaling pathways in CML progenitors but does not induce a strong proapoptotic response. These observations argue against a prominent role for Src kinases in persistence of primitive CML cells in TKI-treated patients. [Cancer Res 2008;68(23):9624–33]