Human myeloid leukemia cell lines are induced to terminal differentiation into monocyte lineage by 1,25-dihydroxyvitamin D3 (1,25D3) or its analogs (deltanoids). However, translation of these findings to the clinic is limited by calcemic effects of deltanoids. Strategies to overcome this problem include combination of deltanoids with other compounds to induce differentiation at lower, noncalcemic, deltanoid concentrations. We previously showed that either carnosic acid, an antioxidant, or SB202190, a p38 MAPK inhibitor, increase the potency of 1,25D3 in the HL60 cell line. Here, we report that simultaneous addition of both these agents further increases differentiation potency of deltanoids in this cell line and in freshly obtained leukemic cells ex vivo. Activity of MAPK pathways showed that increased differentiation was associated with enhanced activity of JNK pathway in all responding cell subtypes. Our studies suggest that patients with CML or AML subtypes M2 and M4, but not M1, M3 or M4eo, are particularly suitable for this combination therapy. We conclude that the established cell line HL60 presents a good model for some, but not all, subtypes of myeloid leukemia, and that the JNK pathway plays an important role in monocytic differentiation of human leukemic cells ex vivo, as well as in vitro.