Abstract Cholesterol metabolism is particularly active in malignant, proliferative cells, whereas cholesterol starvation has been shown to inhibit cell proliferation. Inhibition of enzymes involved in cholesterol biosynthesis at steps before the formation of 7-dehydrocholesterol has been shown to selectively affect cell cycle progression from G2 phase in human promyelocytic HL-60 cells. In the present work, we explored whether cholesterol starvation by culture in cholesterol-free medium and treatment with different distal cholesterol biosynthesis inhibitors induces differentiation of HL-60 cells. Treatment with SKF 104976, an inhibitor of lanosterol 14-α demethylase, or with zaragozic acid, which inhibits squalene synthase, caused morphologic changes alongside respiratory burst activity and expression of cluster of differentiation antigen 11c (CD11c) but not cluster of differentiation antigen 14. These effects were comparable to those produced by all-trans retinoic acid, which induces HL-60 cells to differentiate following a granulocyte lineage. In contrast, they differed from those produced by vitamin D3, which promotes monocyte differentiation. The specificity of the response was confirmed by addition of cholesterol to the culture medium. Treatment with PD 98059, an inhibitor of extracellular signal–regulated kinase, abolished both the activation of NADPH oxidase and the expression of the CD11c marker. In sharp contrast, BM 15766, which inhibits sterol Δ7-reductase, failed to induce differentiation or arrest cell proliferation. These results show that changes in the sterol composition may trigger a differentiation response and highlight the potential of cholesterol pathway inhibition as a possible tool for use in cancer therapy. [Cancer Res 2007;67(7):3379–86]