Chronic myelogenous leukemia (CML) results from malignant transformation of a primitive hematopoietic cell by the BCR/ABL oncogene. The breakpoint cluster region/ABL (BCR/ABL) tyrosine kinase inhibitor imatinib mesylate (imatinib) is highly effective in inducing remissions in CML. However, the effects of imatinib on intracellular signaling in primary progenitor cells are not well described. We show that imatinib exposure resulted in a significant dose-responsive reduction in BCR/ABL kinase activity in CML CD34⁺ cells. However, imatinib treatment resulted in an increase in activity of p42/44 mitogen-activated protein kinase (MAPK), an important downstream effector of BCR/ABL. Increased MAPK activity was growth factor dependent. Pharmacologic inhibition of MAPK using MAPK/extracellular signal–regulated kinase kinase–1/2 (MEK-1/2) inhibitors significantly reduced CML progenitor proliferation. Combined treatment with a MEK-1/2 inhibitor and imatinib significantly increased suppression of CML progenitors compared with either inhibitor alone. In contrast, imatinib treatment resulted in a small reduction in AKT activity. Combined treatment with a phosphatidylinositol-3 (PI-3) kinase inhibitor and imatinib significantly increased suppression of CML progenitor growth compared with either inhibitor alone. We conclude that inhibition of BCR/ABL kinase activity in CML progenitors by imatinib results in a growth factor-dependent compensatory increase in MAPK activity and in only partial inhibition of PI-3 kinase activity. These mechanisms may contribute to incomplete elimination of CML progenitors by imatinib. (Blood. 2004;103:3167-3174)