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 sp red fluorescent protein (DsRed) reporter gene transfer in committed hematopoietic progenitors compared with both the early-generation hyperactive SB11 transposase and the piggyBac transposon 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 IL;cnull (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 revealedSBtransposon sequences in thehumanchromosomes 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 humanhematopoietic stem cells as a modality for gene therapy.