Three-color flow cytometry was used to analyze the coexpression of surface antigens on megakaryocytes (MKs) developing in liquid cultures of enriched CD34+ cells purified from cord blood. Cells were cultured in serum-replete medium supplemented with interleukin 3 (IL-3), stem cell factor and IL-6. During two weeks of culture, total cells increased 76 +/- 36-fold. CD34+ cells maximally expanded between days 2 and 4, and then gradually decreased to their original input numbers by day 14. As CD34+ cells declined, MKs, defined as glycoprotein (GP) IIbIIIa+ cells, steadily increased in culture 20.9 +/- 18.3-fold. Megakaryopoiesis was further defined by monitoring the expression of GPs IIb, IIIa, Ib, IbIX, and IIIb and c-kit antigen. Increased expression of GPs IIbIIIa and IIb occurred earliest in culture, followed by IIIa and Ib, and then IbIX. Expression of IIIb, also found on monocytes, did not parallel that of the other antigens except when coexpressed on IIbIIIa+ cells. c-kit expression paralleled that of CD34 until the second week of culture when expression was high on nonMKs. Each of these antigens was coexpressed on CD34+ cells and identified a subset of late MK progenitors that increased steadily in culture. Triplelabeled cells expressing CD34, IIbIIIa and a third MK-related antigen were seen at all times. Polyploid MKs of up to 32N were observed during the second week of culture. Multiparametric flow cytometry proved to be a rapid, sensitive and specific method for quantitating the changes in antigen expression of differentiating MKs.