Here we report stable gene transfer in cord blood-derived CD34+hematopoietic stem cells using a hyperactive nonviral Sleeping Beauty(SB) transposase (SB100X). In colony-forming assays, SB100X mediated the highest efficiency (24%) of stable Discosoma spred fluorescent protein (DsRed) reporter gene transfer in committed hematopoietic progenitors compared with both the early-generation hyperactive SB11 transposase and the piggyBactransposon system (1.23% and 3.8%, respectively). In vitro differentiation assays further demonstrated that SB100X-transfected CD34+cells can develop into DsRed+CD4+CD8+T (3.17%-21.84%; median, 7.97%), CD19+B (3.83%-18.66%; median, 7.84%), CD56+CD3−NK (3.53%-79.98%; median, 7.88%), and CD33+myeloid (7.59%-15.63%; median, 9.48%) cells. SB100X-transfected CD34+cells achieved approximately 46% engraftment in NOD-scid IL2γcnull(NOG) mice. Twelve weeks after transplantation, 0.57% to 28.96% (median, 2.79%) and 0.49% to 34.50% (median, 5.59%) of total human CD45+cells in the bone marrow and spleen expressed DsRed, including CD19+B, CD14+monocytoid, and CD33+myeloid cell lineages. Integration site analysis revealed SB transposon sequences in the human chromosomes of in vitro differentiated T, B, NK, and myeloid cells, as well as in human CD45+cells isolated from bone marrow and spleen of transplanted NOG mice. Our results support the continuing development of SB-based gene transfer into human hematopoietic stem cells as a modality for gene therapy.