Cell treatment with (2-Hydroxypropyl)-β-cyclodextrin results in the activation of the transcription factor EB, a master regulator of lysosomal function and autophagy, and in enhancement of the cellular autophagic clearance capacity. (2-Hydroxypropyl)-β-cyclodextrin treatment reduces intracellular cholesterol resulting in significant leukemic cell growth inhibition through G2/M cell-cycle arrest and apoptosis. The IC50 values for (2-Hydroxypropyl)-β-cyclodextrin after 72 hours exposure are in the range of 3.86–10.09 mM. (2-Hydroxypropyl)-β-cyclodextrin also shows anticancer effects against CML cells expressing a T315I BCR-ABL mutation (that confers resistance to most ABL tyrosine kinase inhibitors), and hypoxia-adapted CML cells that have characteristics of leukemic stem cells. In addition, colony forming ability of human primary AML and CML cells is inhibited by (2-Hydroxypropyl)-β-cyclodextrin.