Erythropoietin (EPO) binds its specific receptor (EPO-R) to mediate survival and proliferation of definitive (bone marrow/fetal liver) red cell precursors. In contrast, little is known about cytokine signaling inprimitive (yolk sac) erythroblasts. We detected EPO-R mRNA by in situ hybridization at early stages of murine yolk sac blood island development(E7.5-E8.5). Because the size and location of the mammalian embryo makes experimental manipulation difficult, we developed a serum-free murine yolk sac explant culture system {Blood 86:156, 1995}, and an antisense experimental approach to examine the functional role of EPO/EPO-R during the initial development of primary primitive erythroblasts from extra-embryonic mesoderm cells. The addition of EPO to E7.5 yolk sac explants increased both the accumulation of βH1-globin mRNA and the number of differentiating yolk sac erythroblasts compared to untreated explants. Antisense EPO-R ODN decreased βH1-globin accumulation 95% (p<0.005), the number of differentiating primitive erythroblasts 56% (p<0.01), and the number of erythroid progenitors (CFU-E and BFU-E) > 50% (p<0.05), compared to missense EPO-R ODN-treated and untreated explants. A similar experimental approach was used to investigate putative EPO-R intracellular signaling cascades. Antisense c-raf and Jak2 ODN each decreased the number of primitive erythroblasts 40% (p<0.01), but only antisense c-raf ODN significantly decreased erythroid progenitors. We conclude that EPO/EPO-R signaling is functionally active during the initial differentiation of primary yolk sac erythroid cells, and suggest that this signaling may involve both the ras/raf and the Jak2/Stat pathways.