In vivo selection of hematopoietic stem cells (HSCs) offers an approach to enrichment of genetically modified blood cells in the context of gene therapy for blood disorders. We have previously demonstrated efficient HSC selection in mice using retroviral vectors expressing dihydrofolate reductase (DHFR) or methylguanine methyltransferase (MGMT) drug resistance genes. In this study, we examined whether drug selection was followed by subsequent HSC regeneration and, if not, whether regeneration could be augmented by enforced expression of HOXB4, which has previously been shown to enhance HSC regeneration after transplant. Using a murine competitive repopulation model, we found that selection using either the DHFR or the MGMT system was accompanied by a significant overall reduction in repopulating activity in secondary transplant assays, although hematopoiesis remained normal after recovery. Inclusion of a HOXB4 expression cassette in the DHFR vector resulted in a partial restoration of HSC numbers following selection and was associated with an increase in HSC selection efficiency. These results illustrate that while drug resistance vectors can protect transduced HSC from cytotoxic drugs, the self-renewal capacity of transduced HSCs is limited following in vivo selection. Strategies that increase self-renewal capacity could increase the efficiency and safety of in vivo selection.