Vitamin D derivatives can induce differentiation of human acute myeloid leukemia (AML) cells. Here, we investigated if the G₁ cell cycle block associated with monocytic differentiation is modulated by the p53 status of the cells treated with 1,25D, alone or with plant antioxidants carnosic acid (C) or silibinin (S), and a p38 MAPK inhibitor SB202190 (SB), a combination (D-C/S-SB) previously shown to enhance differentiation of AML p53null cells. D-C/S-SB enhanced differentiation of OCI-AML3 (p53wt) and as expected HL60 (p53 null) cells, but not of MOLM-13 (p53wt) cells. Conversely, MOLM-13 (p53wt) cells treated with 1,25D and/or D-C/S-SB, resembled HL60 (p53 null) cells in rapid G₁ block, while OCI-AML3 (p53wt) cells showed a delayed G₁ block when treated in a similar way, indicating that there is no relationship between the p53 status and G₁ block. Western blot analysis revealed that 1,25D and D-C/S-SB increased the inhibitory phosphorylation levels MEK-1 (P-Thr286), but decreased the levels of activated ERK1/2 (Thr202/Tyr204;Thr185/Tyr187), again without any apparent relationship to the p53 status. Interestingly, the increased levels of p21(Waf1/Cip1) were insufficient to promote a G₁ block in this system, as only cell lines with increased levels of p27(Kip1) and p35Nck5a, an activator of Cdk5, showed a rapid G₁ block. Overall, our data show that the p53-p21 axis is unlikely to have a role in differentiation-associated G₁ block in AML cells with wt p53, and that this block is achieved by several, possibly co-operating but redundant pathways, that include inhibition of MEK-1 by p35Nck5a-activated Cdk5.