Though both low‐speed centrifugation and the use of fibronectin (Retronectin) fragments increase gene transduction efficiency, they still do not overcome the adverse effects of the presence of virus‐containing medium (VCM). In this study, we improved transduction efficiency of primitive human hematopoietic cells by optimizing the conditions for preadsorbing culture dishes with retrovirus using a centrifugation protocol allowing subsequent infection to be carried out in the absence of VCM. We also demonstrate that preadsorbing tissue culture plates with retrovirus is dependent on the volume of VCM used for preadsorption and the length of centrifugation and the type of plasticware used but not on the temperature of centrifugation (4–33 °C). Direct exposure of CD34⁺ target cells to VCM depletes the primitive CD34⁺CD38^neg subpopulation by more than 30%, whereas the optimized VCM‐free infection protocol targets this population with equivalent efficiency but had no detrimental effects on CD34⁺CD38^neg frequency. In summary, we demonstrate a high‐frequency transduction protocol which preserves the therapeutically relevant primitive subpopulation of human hematopoietic cells.