We developed a new in vivo hematopoietic stem cell (HSC) gene therapy approach that involves only IV injections and does not require myeloablation/conditioning and HSC transplantation. In this approach, HSCs are mobilized from the bone marrow into the peripheral bloodstream and transduced with IV injected helper-dependent adenovirus (HDAd) vectors. A fraction of transduced HSCs returns to the bone marrow and persists there long term. Here, we report desmoglein 2 (DSG2) as a new receptor that can be used for in vivo HSC transduction. HDAd5/3+ vectors were developed that use DSG2 as a high-affinity attachment receptor, and in vivo HSC transduction and safety after IV injection of an HDAd5/3+ vector expressing green fluorescent protein (GFP) in granulocyte colony-stimulating factor/AMD3100 (plerixafor)-mobilized rhesus macaques were studied. Unlike previously used CD46-targeting HDAd5/35++ vectors, HDAd5/3+ virions were not sequestered by rhesus erythrocytes and therefore mediated ∼10-fold higher GFP marking rates in primitive HSCs (CD34+/CD45RA–/CD90+ cells) in the bone marrow at day 7 after vector injection. To further increase the return of in vivo transduced, mobilized HSCs to the bone marrow, we transiently expressed cxcr4 in mobilized HSCs from the HDAd5/3+ vector. In vivo transduction with an HDAd5/3+GFP/cxcr4 vector at a low dose of 0.4 × 1012 viral particles/kg resulted in up to 7% of GFP-positive CD34+/CD45RA–/CD90+ cells in the bone marrow. This transduction rate is a solid basis for in vivo base or prime editing in combination with natural or drug-induced expansion of edited HSCs. Furthermore, our study provides new insights into HSC biology and trafficking after mobilization in nonhuman primates. •A DSG2-targeting HDAd5/3+ vector efficiently transduced mobilized HSCs in vivo in rhesus macaques.•Transient overexpression of cxcr4 in mobilized, in vivo transduced HSCs greatly increased their return to the bone marrow. Display omitted