Erythropoietin (Epo) is a cytokine known to stimulate proliferation and differentiation of erythroid cells. However, recent gene disruption experiments demonstrated that Epo receptor signaling is not an obligatory step in erythroid differentiation. Here, we describe the role of Epo in proliferation, terminal differentiation, and apoptosis in a novel human Epo-dependent cell line, AS-E2. Upon withdrawal of Epo, the cells ceased to proliferate and underwent apoptotic death. Accompanying this cell death, an increase in the number of hemoglobin-positive cells of approximately 2-fold was observed. This was associated with immediate up-regulation of the GATA-1:GATA-2 ratio and down-regulation of Bcl-xL. Treatment with Epo or 12-O-tetradecanoyl-phorbol-13-acetate (TPA) up-regulated expression of GATA-2 and Bcl-xL, and these elevations were inhibited by inhibitors of protein kinase C (PKC), H7 and H8. HA1004, a structural analogue of H7 but a poor inhibitor of PKC, had no inhibitory effect. Therefore, in AS-E2 cells, it is likely that Epo plays a role in (a) proliferation, (b) inhibition of differentiation, and (c) survival, by maintaining GATA-2 and Bcl-xL expression through activation of PKC.