Chronic myeloid leukemia (CML) is a hematopoietic stem/progenitor cell disorder in which Bcr-Abl oncoprotein inhibits cell differentiation. Differentiation induction is currently considered an alternative strategy for treating CML. Activin A, a member of the transforming growth factor-β superfamily, induces erythroid differentiation of CML cells through the p38 mitogen-activated protein kinase (MAPK) pathway. In this study, pretreatment of K562 CML stem/progenitor cell line with activin A followed by a subtoxic concentration of Bcr-Abl inhibitor imatinib strongly induced growth inhibition and apoptosis compared to the simultaneous co-treatment scheme. Imatinib induced growth inhibition and apoptosis following activin A pretreatment were dose- and time-dependent manners. Imatinib-induced growth inhibition and apoptosis was also dependent on dosing of activin A pretreatment. More than 90% of the activin A-induced increases of glycophorin A-positive cells were sensitive to imatinib. However, only the part of original glycophorin A-positive cells after activin A treatment were sensitive to imatinib. Activin A/imatinib sequential treatment decreased Bcr-Abl, procaspase-3, Mcl-1 and Bcl-xL. This treatment also induced cleavage of procaspase-3/PARP. The reduction of erythroid differentiation in p38 MAPK dominant negative mutants or by shRNA knockdown of p38 MAPK decreased the sequential treatment-mediated growth inhibition and apoptosis. Furthermore, the same inhibition level of MDR1 expression was observed under activin A alone, activin A/imatinib sequential treatment or co-treatment. The p38 MAPK inhibitor SB203580 can restore activin A-inhibited MDR1 expression. Taken together, our results suggest that a subtoxic concentration of imatinib could exhibit strong cytotoxicity against erythroid differentiated K562 CML cells.