Humans subjected to periods of microgravity develop anemia, thrombocytopenia, and abnormalities in red blood cell structure. The causes of these abnormalities are complex and unclear. The in vitro effects of spaceflight on hematopoietic cell proliferation and differentiation were investigated during the space shuttle missions STS-63 (Discovery) and STS-69 (Endeavour). CD34+ bone marrow progenitor cells were cultured in liquid suspension culture and on hematopoietic supportive stromal cells using hollow-fiber culture modules. One set of cultures was maintained at microgravity (flight cultures) for the last 8-10 days of culture and a second control was at full gravity (ground control). Over the 11- to 13-test-day period, ground control culture total cell number increased 41.0- to 65.5-fold but flight culture total cell number increased only 10.1- to 17.6-fold (57-84% decrease). Comparing ground control cultures and microgravity cultures, respectively, for progenitor cell content, myeloid progenitor cell numbers expanded 2.6- to 17.5-fold compared with 0.9- to 7.0-fold and erythroid progenitor cell numbers expanded 2.0- to 4.1-fold in ground control cultures but actually declined at microgravity (>83% reduction). Moreover, microgravity cultures demonstrated accelerated maturation/differentiation toward the macrophage lineage. These data indicate that spaceflight has a direct effect on hematopoietic progenitor cell proliferation and differentiation and that specific aspects of in vitro hematopoiesis, particularly erythropoiesis, involve gravity-sensitive components.