Abstract The ATP-binding cassette transporter, ABCG2, is a molecular determinant of the side population phenotype, which is enriched for stem and progenitor cells in various nonhematopoietic and hematopoietic tissues. ABCG2 is highly expressed in hematopoietic progenitors and silenced in differentiated hematopoietic cells, suggesting a role of ABCG2 in early hematopoiesis. To test whether ABCG2 is involved in human hematopoietic development, we retrovirally transduced umbilical cord blood-derived early hematopoietic cells and analyzed hematopoiesis in vitro and in vivo. ABCG2 increased the number of clonogenic progenitors in vitro, including the most primitive colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte, by twofold (n = 14; p < .0005). Furthermore, ABCG2 induced a threefold increase in the replating capacity of primary colonies (n = 9; p < .01). In addition, ABCG2 impaired the development of CD19+ lymphoid cells in vitro. In transplanted NOD/SCID mice, the ATP-binding cassette transporter decreased the number of human B-lymphoid cells, resulting in an inversion of the lymphoid/myeloid ratio. ABCG2 enhanced the proportion of CD34+ progenitor cells in vivo (n = 4; p < .05) and enhanced the most primitive human progenitor pool, as determined by limiting dilution competitive repopulating unit assay (p < .034). Our data characterize ABCG2 as a regulatory protein of early human hematopoietic development. Disclosure of potential conflicts of interest is found at the end of this article.