Abstract Achieving high-level expansion of hematopoietic stem cells (HSCs) in vitro will have an important clinical impact in addition to enabling elucidation of their regulation. Here, we couple the ability of engineered NUP98-HOXA10hd expression to stimulate > 1000-fold net expansions of murine HSCs in 10-day cultures initiated with bulk lin−Sca-1+c-kit+ cells, with strategies to purify fetal and adult HSCs and analyze their expansion clonally. We find that NUP98-HOXA10hd stimulates comparable expansions of HSCs from both sources at ∼ 60% to 90% unit efficiency in cultures initiated with single cells. Clonally expanded HSCs consistently show balanced long-term contributions to the lymphoid and myeloid lineages without evidence of leukemogenic activity. Although effects on fetal and adult HSCs were indistinguishable, NUP98-HOXA10hd–transduced adult HSCs did not thereby gain a competitive advantage in vivo over freshly isolated fetal HSCs. Live-cell image tracking of single transduced HSCs cultured in a microfluidic device indicates that NUP98-HOXA10hd does not affect their proliferation kinetics, and flow cytometry confirmed the phenotype of normal proliferating HSCs and allowed reisolation of large numbers of expanded HSCs at a purity of 25%. These findings point to the effects of NUP98-HOXA10hd on HSCs in vitro being mediated by promoting self-renewal and set the stage for further dissection of this process.