Erythropoiesis generates two million RBCs per second, a robust process sensitive to diet, disease, and genetic disruptions resulting in anemia and supply and safety issues with transfusable blood. Gaining a better understanding of erythropoiesis by flow cytometry has been challenged by the loss of correlation with critical morphological characteristics and a maturation that lacks discrete changes in its limited immunophenotype, challenges shared with analysis many non-immune cells. Imaging flow cytometry (IFC) has enabled us to apply the quantitative power and analytical tools of flow cytometry to the study of erythropoiesis, including control of enucleation, a key limiting step in the goal of ex vivo generation of transfusable blood. This approach is particularly critical in our work on hematopoietic onset in murine embryos where we have demonstrated alternative hematopoietic stem cell (HSC)-independent sources of erythropoiesis. The first wave of hematopoiesis generates “primitive” erythroblasts well as macrophages and platelets, while a second “erythro-myeloid progenitor” (EMP) wave provides definitive erythroid cells, as well as several types of innate immune cells. Differential globin gene usage in primitive and definitive erythropoiesis has been central to studying the complexity of murine erythropoiesis. Early studies of human embryros also indicate waves of HSC-independent erythropoiesis with discrete globin usage are present. However, confusing intermediate and prolonged mixed globin expression during human ESC/iPSC differentiation has confounded utilization of this system to better understand hematopoietic onset and develop cellular therapies. We are applying IFC combined with RNAflow (Primeflow) to single cell analysis of both lineage and maturation during human ES and iPS cell differentiation. Our preliminary data indicates that there actually are two distinct populations of erythroid cells, consistent with primitive and definitive identities as seen in the mouse, but they exist in shifting proportions with both lineages present as erythroblasts at multiple stages of maturation throughout ESC differentiation.