The treatment of chronic myeloid leukemia (CML) with tyrosine kinase inhibitors (TKIs) has substantially extended patient survival. However, TKIs do not effectively eliminate CML stem cells. In fact, CML stem cells persist and cause relapse in the majority of patients upon discontinuation of the drug treatment. Transcriptomic and proteomic analyses have revealed that p53 and c-Myc play defining roles in CML stem cell survival, suggesting that the dual targeting of p53 and c-Myc may selectively eliminate stem cells in patients with CML. Since the downregulation of c-Myc and then upregulation of p21 (a target gene of p53) are commonly observed during the differentiation of acute myeloid leukemia cells induced by differentiation inducers, we hypothesized that differentiation-inducing agents may be useful in regulating c-Myc and p53 expression in CML cells. In the present study, we demonstrate that some differentiation-inducing agents effectively suppress the self-renewal ability of CML cells, and that the combination of these inducers with TKIs results in significantly greater inhibitory effects on CML cell growth compared to the use of TKIs or the inducer alone. The KU812 cells were treated with various concentrations of the inducers in the presence or absence of 30 nM imatinib for 4 days. Among the differentiation inducers we tested, cotylenin A (CN-A) was the most potent at inhibiting the self-renewal ability of the CML cells. CN-A induced the robust expression of CD38, a marker of committed progenitor and more differentiated myelomonocytic cells, and rapidly suppressed c-Myc expression and upregulated p21 expression in CML cells. Thus, these results suggest that CN-A may have potential to promote the elimination of stem cells in CML.