Background: Human hematopoietic stem cells (HSCs) are used for autologous and allogeneic stem cell transplantation. Currently, HSCs serve as target cells for tissue engineering, gene transfer, and gene therapeutic approaches. However, viral transfection systems involve the risk of tumor induction by unspecific genomic integration, whereas non-viral transfection systems often fail due to low transfection efficiency. Material and Methods: CD34-positive peripheral blood stem cells (PBSCs) and bone marrow stem cells (BMSCs) were prepared by immunomagnetic selection with a purity > 99% for PBSCs and > 95% for BMSCs. Cells were transfected with the truncated low-affinity nerve growth factor receptor (deltaLNGFR) using the nucleofection technique. Transfection efficiency and kinetics were measured by FACS analysis, immunohistochemistry, immunofluorescence microscopy, and RT-PCR. Real-time PCR and Southern blot analysis were performed to detect the number of plasmids per cell during long-time cell culture and genomic integration of the transgene, respectively. Results: High transfection efficiency up to 45% for PBSCs and up to 36% for BMSCs was achieved without a marked decrease of cell viability. DeltaLNGFR expression decreased on proliferating HSCs with time, but the marker gene was significantly expressed over a 200-hour time period. No genomic integration of the transgene was observed. Conclusions: Nucleofection is a highly efficient, nontoxic method for transient genetic labeling of HSCs. Transient transfection of HSCs with the marker gene deltaLNGFR is rapid, specific and shows no relevant cell toxicity. This method is useful for in vitro protocols but also for in vivo marker studies in humans.