Lentivirus-based vectors have the potential to transduce non-dividing primary stem cells. However, primary cells tend to be less susceptible to manipulation and require a high concentration of virus inoculum. Furthermore, increasing the concentration of the lentivirus inoculum may raise safety risks. Therefore, to develop a technique that allows high transduction efficiency at low multiplicities of infection (MOIs), we optimized a lentivirus-based system for cell lines and primary cells by determining the best condition using various parameters. When progenitor cell assays were conducted using human CD34(+) bone marrow and mononuclear cells, the transduction condition yielded a great number of eGFP(+) colonies with lower-dose viral inocula compared to that of current lentivirus-based transduction technologies. In conclusion, this system is anticipated to produce stable expression of a gene introduced into primary cells for preclinical studies with lower safety risks.