Ex vivo expansion of hematopoietic stem cells (HSCs) has been explored in the fields of stem cell biology, gene therapy, and clinical transplantation. Here, we demonstrate efficient ex vivo expansion of HSCs measured by long‐term severe combined immunodeficient (SCID) repopulating cells (SRCs) from human cord blood CD133‐sorted cells using a soluble form of Delta1. After a 3‐week culture on immobilized Delta1 supplemented with stem cell factor, thrombopoietin, Flt‐3 ligand, interleukin (IL)‐3, and IL‐6/soluble IL‐6 receptor chimeric protein (FP6) in a serum‐ and stromal cell‐free condition, we achieved approximately sixfold expansion of SRCs when evaluated by limiting dilution/transplantation assays. The maintenance of full multipotency and self‐renewal capacity during culture was confirmed by transplantation to nonobese diabetic/SCID/γc^null mice, which showed myeloid, B, T, and natural killer cells as well as CD133⁺CD34⁺ cells, and hematopoietic reconstitution in the secondary recipients. Interestingly, the CD133‐sorted cells contained approximately 4.5 times more SRCs than the CD34‐sorted cells. The present study provides a promising method to expand HSCs and encourages future trials on clinical transplantation.