Clinically successful hematopoietic cell transplantation is dependent on hematopoietic stem and progenitor cells. Here we identify the matricellular protein Nephroblastoma Overexpressed (Nov, CCN3) as being essential for their functional integrity. Nov expression is restricted to the primitive (CD34) compartments of umbilical vein cord blood, and its knockdown in these cells by lentivirus-mediated RNA interference abrogates their function in vitro and in vivo. Conversely, forced expression of Nov and addition of recombinant Nov protein both enhance primitive stem and/or progenitor activity. Taken together, our results identify Nov (CCN3) as a regulator of human hematopoietic stem or progenitor cells.