Post transplant infusion of donor-type natural killer (NK) cells has been shown to have an anti-leukemia-enhancing effect without evoking GVHD in murine hematopoietic cell transplantation (HCT) models. Here, we tested 14 patients (age, 23–65 years), 12 with acute leukemia and 2 with myelodysplastic syndrome, who underwent HLA-mismatched HCT and subsequently received donor NK cell infusions. Cell donors (age, 16–51 years), comprising seven siblings, five offspring, and two mothers of the patients, underwent growth factor-mobilized leukapheresis for 3–5 days. Cells collected on the first 2–4 days were used for HCT, whereas those collected on the last day were CD34 selected by magnetic-activated cell sorting (median, 2.22 × 106 cells/kg; range, 0.29–5.66). Donor NK cells were generated from the CD34+ cells by ex vivo cell culture over a 6-week period (median, 9.28 × 106 cells/kg; range, 0.33–24.50; CD122/CD56+ 64%; CD3+ 1.0%; and viability 88%). There were no signs of acute toxicity in patients infused with these cells 6–7 weeks post transplant. Overall, one and five patients developed acute and chronic GVHD during post transplant period, respectively. These results showed that clinical-grade donor NK cell production from CD34+ cells is feasible. [ABSTRACT FROM AUTHOR]