Uncovering the cellular and molecular mechanisms by which hematopoietic stem cell (HSC) self-renewal is regulated can lead to the development of new strategies for promoting ex vivo HSC expansion. Here, we report the discovery that alternative (M2)-polarized macrophages (M2-M Phi s) promote, but classical (M1)-polarized macrophages (M1-M Phi s) inhibit, the self-renewal and expansion of HSCs from mouse bone marrow (BM) in vitro. The opposite effects of M1-M Phi s and M2-M Phi s on mouse BM HSCs were attributed to their differential expression of nitric oxide synthase 2 (NOS2) and arginase 1 (Argi), because genetic knockout of Nos2 and Argl or inhibition of these enzymes with a specific inhibitor abrogated the differential effects of M1-M Phi s and M2-M Phi s. The opposite effects of M1-M Phi s and M2-M Phi s on HSCs from human umbilical cord blood (hUCB) were also observed when hUCB CD34(+) cells were cocultured with M1-M Phi s and M2-M Phi s generated from hUCB CD34(-) cells. Importantly, coculture of hUCB CD34(+) cells with human M2-M Phi s for 8 days resulted in 28.7- and 6.6-fold increases in the number of CD34(+) cells and long-term SCID mice-repopulating cells, respectively, compared with uncultured hUCB CD34(+) cells. Our findings could lead to the development of new strategies to promote ex vivo hUCB HSC expansion to improve the clinical utility and outcome of hUCB HSC transplantation and may provide new insights into the pathogenesis of hematological dysfunctions associated with infection and inflammation that can lead to differential macrophage polarization.