Clinical and preclinical applications of human hematopoietic stem cells (HSCs) are often limited by scarcity of cells. Expanding human HSCs to increase their numbers while maintaining their stem cell properties has therefore become an important area of research. Here, we report a robust HSC coculture system wherein cord blood CD34 + CD133 + cells were cocultured with mesenchymal stem cells engineered to express angiopoietin-like-5 in a defined medium. After 11 days of culture, SCID repopulating cells were expanded ∼60-fold by limiting dilution assay in NOD-scid Il2rg -/- (NSG) mice. The cultured CD34 + CD133 + cells had similar engraftment potential to uncultured CD34 + CD133 + cells in competitive repopulation assays and were capable of efficient secondary reconstitution. Further, the expanded cells supported a robust multilineage reconstitution of human blood cells in NSG recipient mice, including a more efficient T-cell reconstitution. These results demonstrate that the expanded CD34 + CD133 + cells maintain both short-term and long-term HSC activities. To our knowledge, this ∼60-fold expansion of SCID repopulating cells is the best expansion of human HSCs reported to date. Further development of this coculture method for expanding human HSCs for clinical and preclinical applications is therefore warranted.