Vitamin D3 (VD3) induces monocytic differentiation of U937 cells. Induction of p21Cip1/WAF1 (p21) and subsequent G0/G1 cell-cycle arrest are required in this process. Using a system of inducible expression of ectopic p21, we demonstrated the important role of p21 in the induction of monocytic differentiation in U937 cells. Prior induction of antisense-p21 expression significantly suppressed p21 expression, and resulted in inhibition of VD3-induced U937 differentiation. Moreover, induction of expression of antisense-p21 in VD3-differentiated U937 cells resulted in apoptosis of the cells. This was associated with activation of Cdc2 and caspase-3 like protease. Our results suggest that p21 is required for the initiation of the early steps of differentiation as well as survival of differentiated cells.