Expansion and/or maintenance of hematopoietic stem cell (HSC) potential following in vitro culture remains a major obstacle in stem cell biology and bone marrow (BM) transplantation. Several studies suggest that culture of mammalian cells in microgravity (μ-g) may reduce proliferation and differentiation of these cells. We investigated the application of these findings to the field of stem cell biology in the hopes of expanding HSC with minimal loss of hematopoietic function. To this end, BM CD34 cells were cultured for 4–6 d in rotating wall vessels for simulation of μ-g, and assessed for expansion, cell cycle activation, apoptosis, and hematopoietic potential. While CD34 cells cultured in normal gravity (1-g) proliferated up to threefold by day 4–6, cells cultured in μ-g did not increase in number. As a possible explanation for this, cells cultured in simulated μ-g were found to exit G₀/G₁ phase of cell cycle at a slower rate than 1-g controls. When assayed for primitive hematopoietic potential in secondary conventional 1-g long-term cultures, cells from initial μ-g cultures produced greater numbers of cells and progenitors, and for a longer period of time, than cultures initiated with 1-g control cells. Similar low levels of apoptosis and adhesion molecule phenotype in μ-g and 1-g–cultured cells suggested similar growth patterns in the two settings. These data begin to elucidate the effects of μ-g on proliferation of human hematopoietic cells and may be potentially beneficial to the fields of stem cell biology and somatic gene therapy.