Burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) cells are erythroid progenitors traditionally defined by colony assays. We developed a flow cytometry-based strategy for isolating human BFU-E and CFU-E cells based on the changes in expression of cell surface markers during in vitro erythroid cell culture. BFU-E and CFU-E are characterized by CD45+GPA-IL-3R-CD34+CD36-CD71low and CD45+GPA-IL-3R-CD34-CD36+CD71high phenotypes, respectively. Colony assays validated phenotypic assignment giving rise to BFU-E and CFU-E colonies, both at a purity ~90%. The BFU-E colony forming ability of CD45+GPA-IL-3R-CD34+CD36-CD71low cells required SCF and erythropoietin, while the CFU-E colony forming ability of CD45+GPA-IL-3R-CD34-CD36+CD71high cells required only erythropoietin. Bioinformatic analysis of the RNA-seq data revealed unique transcriptomes in each differentiation stage. The sorting strategy was validated in uncultured primary cells isolated from bone marrow and peripheral blood, indicating that marker expression is not an artifact of in vitro cell culture, but represents an in vivo characteristic of erythroid progenitor populations. The ability to isolate highly pure human BFU-E and CFU-E progenitors will enable detailed cellular and molecular characterization of these distinct progenitor populations and define their contribution to disordered erythropoiesis in inherited and acquired hematological disease. Our data provide important resource for future studies. Overall design: Transcription profiles of Human erythroid progenitors at distinct developmental stages were generated by deep sequencing, in triplicate, using IlluminaHiSeq 2000. The complete dataset comprises 4 sample types: CD34, BFU, CFU, and Pro (reanalysis of GSM1304777-GSM1304779).