Differentiation therapy for cancer is a developing treatment modality that is based on the anti-proliferative effects associated with differentiation of the malignant cells. 1,25-dihydroxyvitamin D(3) (1,25D) and its analogs are currently being evaluated clinically, alone or in combination with other agents, for treatment of several neoplastic diseases, but their usefulness as single agents may be limited by the enhancement of cell survival in some cell types exposed to 1,25D. In this study we evaluated the role of AKT signaling pathway, known to be anti-apoptotic in diverse cell types, in enhancing the survival of human leukemia HL60 cells induced to differentiate with 1,25D. We found that the phosphorylation and activity of AKT, as well as of its downstream targets, are increased after the exposure to 1,25D. Treatment of HL60 cells with PI3K inhibitors LY294002 and Wortmannin, which decrease the activity of the AKT pathway, induced apoptosis, but this effect was reduced in cells simultaneously treated with 1,25D. Interestingly, LY294002 and Wortmannin also accentuated the 1,25D-induced G(1) to S phase cell cycle block in HL60 cells, and this was associated with an increased expression of p27Kip1. Thus, a combination of 1,25D with inhibitors of AKT pathway is strongly anti-proliferative and should therefore be considered for differentiation therapy of myeloid leukemia.