The use of lentiviral vectors (LVs) for gene transfer in research, technological, or clinical applications requires the production of large amounts of vector. Mass production of clinical-grade LVs remains a challenge and limits certain perspectives for therapeutic use. Some improvements in LV production protocols have been possible by acting on multiple steps of the production process. The addition of animal-derived cholesterol to the culture medium of producer cells is known to increase the infectivity of LVs. To avoid the use of this animal-derived product in clinical settings, an alternative approach is to increase de novo the production of cholesterol by overexpressing a crucial cholesterogenic enzyme, namely, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR). This project evaluates the impact of such an approach on the production, infectivity, and stability of LVs. We demonstrated that the overexpression of human HMGCR isoform 1 (hHMGCR1) in LV producer cells efficiently increased de novo cholesterol biosynthesis and enhanced by 2- to 3-fold the physical and infectious titers of LVs. We also observed that LVs produced in hHMGCR1-overexpressing cells were comparable in stability to LVs produced under classical conditions and were capable of transducing human CD34+ hematopoietic stem/progenitor cells efficiently. Interestingly, we also showed that LV production in the absence of fetal calf serum (FCS) but under hHMGCR1-overexpressing conditions allowed a viral production yield comparable to that achieved under classical conditions in high FCS content, leading the way to the establishment of new LV production protocols on adherent cells without serum.