Although discovery of the tyrosine kinase inhibitor (TKI) imatinib mesylate has significantly improved the prognosis of chronic myeloid leukemia (CML) patients, a rare population of CML stem cells is known to be resistant to TKI therapy, causing recurrence of CML. However, recent progress in CML stem cell biology may present a novel therapeutic avenue for CML patients. In this review, we focus on mechanisms used by CML stem cells to maintain TKI-resistance. Comprehensive approaches including mouse genetics, prospective identification of CML stem cells, and syngenic transplantation techniques have identified several key molecules or signaling pathways, including hedgehog (Hh)/Smo, promyelocytic leukemia (PML), 5-lipoxygenase (5-LO), and forkhead box class O (FOXO), that function in CML stem cell maintenance. Inhibiting some of these factors in combination with TKI administration successfully antagonized resistance of CML stem cells to TKI therapy, resulting in efficient eradication of leukemia cells in vivo. Thus, development of methods that sensitize CML stem cells to TKI therapy may lead to novel therapies to treat CML patients.