Hematopoietic stem cells (HSCs) have been used therapeutically for decades, yet their widespread clinical use is hampered by the inability to expand HSCs successfully in vitro. In culture, HSCs rapidly differentiate and lose their ability to self-renew. We hypothesize that by mimicking aspects of the bone marrow microenvironment in vitro we can better control the expansion and differentiation of these cells. In this work, derivatives of poly(ethylene glycol) diacrylate hydrogels were used as a culture substrate for hematopoietic stem and progenitor cell (HSPC) populations. Key HSC cytokines, stem cell factor (SCF) and interferon- (IFN), as well as the cell adhesion ligands RGDS and connecting segment 1 were covalently immobilized onto the surface of the hydrogels. With the use of SCF and IFN, we observed significant expansion of HSPCs, approximate to 97 and approximate to 104 fold respectively, while maintaining c-kit(+)lin(-) and c-kit(+)Sca1(+)lin(-) (KSL) populations and the ability to form multilineage colonies after 14 days. HSPCs were also encapsulated within degradable poly(ethylene glycol) hydrogels for three-dimensional culture. After expansion in hydrogels, approximate to 60% of cells were c-kit(+), demonstrating no loss in the proportion of these cells over the 14 day culture period, and approximate to 50% of colonies formed were multilineage, indicating that the cells retained their differentiation potential. The ability to tailor and use this system to support HSC growth could have implications on the future use of HSCs and other blood cell types in a clinical setting. Biotechnol. (c) 2015 Wiley Periodicals, Inc.