Lipid rafts mediate several survival signals in the development of chronic myeloid leukemia (CML). Methyl-β-cyclodextrin (MβCD) is an inhibitor specifically designed to disrupt lipid rafts in cells by depleting the cholesterol component. We hypothesize that treatment of CML cells with MβCD and imatinib could reduce imatinib resistance. Apoptotic and autophagic cell death was assayed using annexin V–propidium iodide double staining, immunoblotting, and immunocytochemistry. We next investigated whether MβCD could enhance the cytotoxicity of imatinib in imatinib-sensitive and imatinib-resistant K562 cells. Extracellular signal-regulated kinase/sphingosine kinase 1 signaling downstream of lipid raft-activated signaling pathways was significantly inhibited by treatment of cells with a combination of MβCD and imatinib compared with treatment with either agent alone. MβCD induces programmed cell death in CML cells, and its antileukemia action is synergistic with that of imatinib.