Abstract“Autonomous” development of erythroid colonies in erythropoietin (EPO)‐free semi‐solid culture has been used as an in vitro assay for diagnosis of polycythemia vera (PV). These colonies, however, are small and poorly hemoglobinized, rendering the assay in many cases unreliable. We report here on the use of a novel assay; it combines a modified culture procedure that maximizes the growth of EPO‐independent erythroid cells, and immunofluorescence flow cytometry for their detection and quantitation. Peripheral blood mononuclear cells are cultured for 2–5 days in the presence of a combination of growth factors. During this phase, early erythroid committed progenitors, burst forming units (BFUe), proliferate and differentiate into colony forming units (CFUe)‐like progenitors. In the second phase, the latter cells, in the presence of stem cell factor, hemin, and iron‐saturated transferrin, continue to proliferate and mature into hemoglobin (Hb)‐containing orthochromatic normoblasts. Neither phases contained EPO. The culture produced large, pure, and synchronized erythroid cell populations. The cells were then dually labeled with fluorescent probes, nuclear DNA with thiazole orange and intracellular hemoglobin (Hb) with phycoerythrin‐conjugated monoclonal antibodies against human Hb. Cells positive for both labels were assigned as Hb‐containing nucleated precursors. The presence of such cells in EPO‐free cultures indicated “autonomous growth.” None of the EPO‐free cultures derived from normal donors or patients with secondary polycythemia contained such cells. Cultures derived from PV patients contained from 5 to 92% “autonomously‐grown” cells. These culture and analysis methods should minimize false negative results with PV patients and provide objective and quantitative data. Am. J. Hematol. 54