Erythropoietin (Epo) is the principal regulator of the erythropoietic response to hypoxic stress, through its receptor, EpoR. The EpoR signals mediating the stress response are largely unknown, and the spectrum of progenitors that are stress responsive is not fully defined. Here, we used flow cytometry to identify stress-responsive Ter119+CD71highFSChigh early erythroblast subsets in vivo. In the mouse spleen, an erythropoietic reserve organ, early erythroblasts were present at lower frequencies and were undergoing higher rates of apoptosis than equivalent cells in bone marrow. A high proportion of splenic early erythroblasts coexpressed the death receptor Fas, and its ligand, FasL. Fas-positive early erythroblasts were significantly more likely to coexpress annexin V than equivalent, Fas-negative cells, suggesting that Fas mediates early erythroblast apoptosis in vivo. We examined several mouse models of erythropoietic stress, including erythrocytosis and beta-thalassemia. We found a dramatic increase in the frequency of splenic early erythroblasts that correlated with down-regulation of Fas and FasL from their cell surface. Further, a single injection of Epo specifically suppressed early erythroblast Fas and FasL mRNA and cell-surface expression. Therefore, Fas and FasL are negative regulators of erythropoiesis. Epo-mediated suppression of erythroblast Fas and FasL is a novel stress response pathway that facilitates erythroblast expansion in vivo.