Early natural killer (NK)-cell repopulation after allogeneic stem cell transplantation (allo-SCT) has been associated with reduced relapse rates without an increased risk of graft-versus-host disease, indicating that donor NK cells have specific antileukemic activity. Therefore, adoptive transfer of donor NK cells is an attractive strategy to reduce relapse rates after allo-SCT. Since NK cells of donor origin will not be rejected, multiple NK-cell infusions could be administered in this setting. However, isolation of high numbers of functional NK cells from transplant donors is challenging. Hence, we developed a cytokine-based ex vivo culture protocol to generate high numbers of functional NK cells from granulocyte colony-stimulating factor (G-CSF)-mobilized CD34(+) hematopoietic stem and progenitor cells (HSPCs). In this study, we demonstrate that addition of aryl hydrocarbon receptor antagonist StemRegenin1 (SR1) to our culture protocol potently enhances expansion of CD34(+) HSPCs and induces expression of NK-cell-associated transcription factors promoting NK-cell differentiation. As a result, high numbers of NK cells with an active phenotype can be generated using this culture protocol. These SR1-generated NK cells exert efficient cytolytic activity and interferon-γ production toward acute myeloid leukemia and multiple myeloma cells. Importantly, we observed that NK-cell proliferation and function are not inhibited by cyclosporin A, an immunosuppressive drug often used after allo-SCT. These findings demonstrate that SR1 can be exploited to generate high numbers of functional NK cells from G-CSF-mobilized CD34(+) HSPCs, providing great promise for effective NK-cell-based immunotherapy after allo-SCT.