Improving hematopoietic stem and progenitor cell (HSPC) permissiveness to lentiviral vector (LV) transduction without compromising their biological properties remains critical for broad-range implementation of gene therapy as a treatment option for several inherited diseases. This study demonstrates that the use of one-hit ex vivo LV transduction protocols based on either cyclosporin A (CsA) or rapamycin enable as efficient gene transfer as the current two-hit clinical standard into bone marrow–derived CD34+ cells while better preserving their engraftment capacity in vivo . CsA was additive with another enhancer of transduction, prostaglandin E2, suggesting that tailored enhancer combinations may be applied to overcome multiple blocks to transduction simultaneously in HSPC. Interestingly, besides enhancing LV transduction, CsA also significantly reduced HSPC proliferation, preserving the quiescent G0 fraction and the more primitive multipotent progenitors, thereby yielding the highest engraftment levels in vivo . Importantly, no alterations in the vector integration profiles could be detected between CsA and control transduced HSPC. Overall, the present findings contribute to the development of more efficient and sustainable LV gene therapy protocols, underscoring the benefits of scaling down required vector doses, as well as shortening the HSPC ex vivo culture time.