Hematopoietic stem cells (HSCs) are an important target cell population for gene therapy since they can reconstitute the entire hematopoietic system. HSC-enriched cell populations can be recognized based on cell surface marker expression, such as CD34, which is broadly expressed on immature and partially differentiated cells. In mice, co-expression of CD34 and CD105 was previously shown to be relatively more specific for the most immature, long-term repopulating HSCs. Here, we evaluated whether CD105, which is expressed on 30%-80% of CD34(+) cells, is a marker also for human long-term repopulating HSCs. Therefore, we tracked the mature progeny of CD34(+) cells transduced with the CD105-targeted lentiviral vector CD105-LV in xenotolerant mice. Transduction was blocked with soluble CD105 protein confirming specificity. Importantly, CD105-LV transduced human CD34(+) cells engrafted in NOD-scid IL2Rγ(-/-) mice with up to 20% reporter gene-positive cells detected long term in all human hematopoietic lineages in bone marrow (BM), spleen, and blood. In addition, competitive repopulation experiments in mice showed a superior engraftment of CD105-LV transduced CD34(+) cells in BM and spleen compared with cells transduced with a conventional nontargeted lentiviral vector. Thus, human CD34(+)/CD105(+) cells are enriched for early HSCs with high repopulating capacity. Targeting this cell population with CD105-LV offers a novel gene transfer strategy to reach high engraftment rates of transduced cells and highlights the applicability of receptor-targeted vectors to trace cell subsets offering an alternative to prospective isolation by surface markers.