Blood stem cells, also termed hematopoietic stem cells (HSC), are used clinically to treat numerous blood cancers. However, due to the low numbers of HSCs that can be isolated from the bone marrow of donors, broad application of this treatment is severely hindered. A robust method is needed that enables in vitro propagation and expansion of HSCs. In addition, as soon as HSCs are removed from their bone marrow niches and put in culture, they begin to differentiate and lose their multipotency. Therefore, a different experimental approach is required to control HSC function in vitro. We have developed a novel microfabrication process to generate synthetic hydrogel substrates comprised of arrays of microwells that can be locally functionalized with desired stem cell regulatory proteins in simplified ?artificial niches?. Time-lapse microscopy of single cells in conjunction with image analysis was employed to quantify changes in the proliferation kinetics of individual HSCs in response to specific niche proteins. In combination with subsequent in vivo transplantation assays, we identified proteins that maintain stem cell multipotency in vitro by controlling self-renewal divisions.