Red blood cells (RBCs), which constitute the most abundant cell type in the body, come in two distinct flavors- primitive and definitive. Definitive RBCs in mammals circulate as smaller, anucleate cells during fetal and postnatal life, while primitive RBCs circulate transiently in the early embryo as large, nucleated cells before ultimately enucleating. Both cell types are formed from lineage-committed progenitors that generate a series of morphologically identifiable precursors that enucleate to form mature RBCs. While definitive erythroid precursors mature extravascularly in the fetal liver and postnatal marrow in association with macrophage cells, primitive erythroid precursors mature as a semi-synchronous cohort in the embryonic bloodstream. While the cytoskeletal network is critical for the maintenance of cell shape and the deformability of definitive RBCs, little is known about the components and function of the cytoskeleton in primitive erythroblasts. Erythropoietin (EPO) is a critical regulator of late-stage definitive, but not primitive, erythroid progenitor survival. However, recent studies indicate that EPO regulates multiple aspects of terminal maturation of primitive murine and human erythroid precursors, including cell survival, proliferation, and the rate of terminal maturation. Primitive and definitive erythropoiesis share central transcriptional regulators, including Gata1 and Klf1, but are also characterized by the differential expression and function of other regulators, including myb, Sox6, and Bcl11A. Flow cytometry-based methodologies, developed to purify murine and human stage-specific erythroid precursors, have enabled comparative global gene expression studies and are providing new insights into the biology of erythroid maturation.