Although the involvement of the tumor suppressor gene p53 in normal hematopoiesis is uncertain, it can give rise to differentiation signals in leukemic cells. It is not clear, however, whether differentiation merely is a consequence of the ability of p53 to arrest cell proliferation or whether hitherto unknown molecular mechanisms are responsible for the p53-mediated differentiation. To further explore the role of p53 in leukemic cell differentiation, we investigated whether transforming growth factor beta1 (TGF-beta1), a cytokine involved in cell cycle control at several levels, can cooperate with wild-type p53 to induce differentiation of monoblastic U-937 and erythroleukemic K562 cells. Indeed, wild-type p53-expressing cells were found to be more sensitive to TGF-beta1-induced differentiation than control cells, lending support to the idea that p53 is of importance for differentiation induction of leukemic cells. In addition, it is shown that TGF-beta1 can suppress p53-mediated cell death, thus reinforcing the differentiation response. The cyclin-dependent kinase inhibitor p21 and the retinoblastoma protein (pRb) are downstream effectors of p53-mediated growth arrest. Therefore, the roles for these molecules in p53-mediated differentiation were examined. The p53-dependent signals of differentiation were associated with induction of p21 in both cell lines investigated. However, activation of pRb by induced hypophosphorylation and concomitant decreased growth rate on p53-mediated differentiation was observed only in U-937 cells expressing an inducible, temperature-sensitive form of p53 but not in K562 cells constitutively expressing p53. Thus, our data suggest a role for p53 in the regulation of differentiation in leukemic cells that can be independent of its ability to activate pRb and arrest cell proliferation.