Megakaryocytic and erythroid lineages derive from a common bipotential progenitor and share many transcription factors, most prominently factors of the GATA zinc-finger family. Little is known about transcription factors unique to the megakaryocytic lineage that might program divergence from the erythroid pathway. To identify such factors, we used the K562 system in which megakaryocyte lineage commitment is dependent on sustained extracellular regulatory kinase (ERK) activation and is inhibited by stromal cell contact. During megakaryocytic induction in this system, the myeloid transcription factor RUNX1 underwent up-regulation, dependent on ERK signaling and inhibitable by stromal cell contact. Immunostaining of healthy human bone marrow confirmed a strong expression of RUNX1 and its cofactor, core-binding factor β (CBFβ), in megakaryocytes and a minimal expression in erythroblasts. In primary human hematopoietic progenitor cultures, RUNX1 and CBFβ up-regulation preceded megakaryocytic differentiation, and down-regulation of these factors preceded erythroid differentiation. Functional studies showed cooperation among RUNX1, CBFβ, and GATA-1 in the activation of a megakaryocytic promoter. By contrast, the RUNX1-ETO leukemic fusion protein potently repressed GATA-1–mediated transactivation. These functional interactions correlated with physical interactions observed between GATA-1 and RUNX1 factors. Enforced RUNX1 expression in K562 cells enhanced the induction of the megakaryocytic integrin proteins αIIb and α₂. These results suggest that RUNX1 may participate in the programming of megakaryocytic lineage commitment through functional and physical interactions with GATA transcription factors. By contrast, RUNX1-ETO inhibition of GATA function may constitute a potential mechanism for the blockade of erythroid and megakaryocytic differentiation seen in leukemias with t(8;21).