Abstract: Objective To identify the best transfect conditions for lentiviral vector to transfect CD34⁺ stem cells from human cord blood. Methods CD34⁺ hematopoietic stem cells from human cord blood were transduced with pTRIPdU3-RNAiTALh-EF1a-GFP plasmid expressing GFP by the second generation and third generation lentiviral vector system. The transfect conditions such as the concentration of the virus, polybrene, transfect volume and media, multiplicity of infection (MOI) values, incubating time and centrifugation in 12-well plate at 200×g were tested to obtain optimal transfect conditions. The number of CFU were counted and the types of CFU were identified by light microscope after the transfected cells (non-infected stem cells served as control) were cultured for 14 days at a 37℃, 5% CO₂ incubator. Results The second-generation lentiviral vector plasmid had higher infect rate than the third-generation. The optimal transfect conditions were determined as:fresh sorting CD34⁺ cells, 10⁷TU virus concentration, Polybrene 2 μg/mL in opti-MEM medium, centrifuged at 200×g for 1 h and then co-culture 8 h for cells and virus mixture in one well in flat-bottomed 12-well plate (repeated once). Both infected and non-infected CD34⁺ stem cells developed CFUs with similar numbers and types of colonies after being cultured for 14 days in the cytokine-containing 1:1 liquid medium/semi-solid medium. Conclusion The identified optimal conditions can enable effective lentiviral vector transduction of CD34⁺ without interrupting the differentiation potential of the hematopoietic stem cells.