Lineage-specific transcription factors play crucial roles in the development of hematopoietic cells. In a previous study, it was demonstrated that Ras activation was involved in thrombopoietin-induced megakaryocytic differentiation. In this study, constitutive Ras activation by H-ras(G12V) evoked megakaryocytic maturation of erythroleukemia cell lines F-36P and K562, but not of myeloid cell line 32D cl3 that lacks GATA-1. However, the introduction of GATA-1 led to reprogramming of 32D cl3 toward erythrocytic/megakaryocytic lineage and enabled it to undergo megakaryocytic differentiation in response to H-ras(G12V). In contrast, the overexpression of PU.1 and c-Myb changed the phenotype of K562 from erythroid to myeloid/monocytic lineage and rendered K562 to differentiate into granulocytes and macrophages in response to H-ras(G12V), respectively. In GATA-1-transfected 32D cl3, the endogenous expression of PU.1 and c-Myb was easily detectable, but their activities were reduced severely. Endogenous GATA-1 activities were markedly suppressed in PU.1-transfected and c-myb-transfected K562. As for the mechanisms of these reciprocal inhibitions, GATA-1 and PU.1 were found to associate through their DNA-binding domains and to inhibit the respective DNA-binding activities of each other. In addition, c-Myb bound to GATA-1 and inhibited its DNA-binding activities. Mutant GATA-1 and PU.1 that retained their own transcriptional activities but could not inhibit the reciprocal partner were less effective in changing the lineage phenotype of 32D cl3 and K562. These results suggested that GATA-1 activities may be crucial for Ras-mediated megakaryocytic differentiation and that its activities may be regulated by the direct interaction with other lineage-specific transcription factors such as PU.1 and c-Myb.