The study of human erythropoiesis in health and disease requires a robust culture system that consistently and reliably generates large numbers of immature erythroblasts that can be induced to differentiate synchronously. We describe a culture method modified from Leberbauer et al.(2005) and obtain a homogenous population of erythroblasts from peripheral blood mononuclear cells (PBMC) without prior purification of CD34+ cells. This pure population of immature erythroblasts can be expanded to obtain 4× 10 8 erythroblasts from 1× 10 8 PBMC after 13–14 days in culture. Upon synchronized differentiation, high levels of enucleation (80–90%) and low levels of cell death (< 10%) are achieved. We compared the yield of erythroblasts obtained from PBMC, CD34+ cells or PBMC depleted of CD34+ cells and show that CD34− cells represent the most significant early erythroid progenitor population. This culture system may be particularly useful for investigating the pathophysiology of anemic patients where only small blood volumes are available.