The wall of 12 yolk sacs (YSs) from 17- to 50-day-old human embryos was examined by light, scanning, and transmission electron microscopy to identify the ontogeny of embryonic erythropoiesis. Initial formation of blood island with the generation of erythroid and endothelial cells was seen in the mesenchymal layer in embryos aged 17 days. A network of blood vessels containing abundant erythroblasts was identified in the YS walls of embryos aged ∼24 days. At this age, erythroblasts were also identified within the embryo body. Primitive erythroblasts were the only cells present within the embryo and its YS until the end of week 5. These cells first appeared in the mesenchymal vascular plexus of the YS wall, and were then observed in the liver and other tissues of the embryo. At embryonic week 5, two compartments were identified in the YS wall; a mesodermal one in which blood vessels were formed, and an endodermal compartment in which erythrocytes were present within the endodermal vesicles. Erythrocytes were small non-nucleated cells similar to adult erythrocytes. Transmission electron microscopic observation focused on the endodermal vesicles confirmed the presence of definitive erythrocytes only at such extra vascular location. At this age, there were no definitive erythrocytes detected within the embryo. Erythrocytes started to be identified in embryonic blood vessels from week 7 onward. These findings provide information not previously described about YS erythropoiesis during early human development. Microsc. Res. Tech., 2008. © 2008 Wiley-Liss, Inc.