Onset of definitive erythropoiesis by E11.5. (A) Imaging flow cytometry of E11.5 blood. Definitive RBCs (top) have higher mean levels of Ter119 staining and lighter brightfield intensity then cocirculating primitive erythroid cells (bottom). (B) Percent of total erythroid (Ter119-positive) cells that are definitive in fetal blood are plotted by embryonic day (note log scale). Low but consistent numbers of definitive erythroid cells are seen by E11.5. (C) Median diameter of definitive erythroid cells demonstrates early definitive RBC size is consistent with later fetal RBC size and different from primitive or adult definitive erythroid cells. (D) Imaging flow cytometry demonstrated all stages of definitive erythroblasts in the E11.5 liver. Maturational stages were determined by combined morphologic and fluorescent parameters including cell and nuclear size, RNA (thiazole orange) staining and mean intensity of Ter119 and DNA (DRAQ5) staining. (E) Absolute numbers of staged erythroblasts per liver during fetal development. Inset is a smaller scale plot of E11.5 data. Error bars are SEM, n = 3 or 4.