The ability of natural killer (NK) cells to kill tumor cells without antigen recognition makes them appealing as an adoptive immunotherapy. However, NK cells are not routinely used in the context of leukemic relapse after hematopoietic stem cell transplantation. Patients who experience relapse can be treated with donor lymphocyte infusions (DLI) based on small-cell fractions frozen at the time of transplantation. Since peripheral blood stem cells (PBSCs) are increasingly used as a stem cell source and as a source of cells for DLI, we aimed to evaluate the impact of G-SCF mobilization on NK cell phenotype, subset repartition, and functionality. Immunomagnetically isolated NK cells from healthy donor blood, donor PBSCs, and patient PBSCs were expanded for 14 days with IL-15. The expansion capacity, phenotype, and functions (cytokine secretion and cytotoxicity) of NK cell subsets based on CD56 and CD16 expression were then evaluated. Mobilized sources showed a significant decrease of CD56brightCD16+ NK cells (28 versus 74%), whereas a significant increase (64 versus 15%) of CD56brightCD16- NK cells was observed in comparison with peripheral blood. Patient-mobilized NK cells showed a significantly decreased cytotoxicity, and antibody-dependent cell cytototoxicity (ADCC) was also observed to a lesser extent in NK cells from healthy donor PBSC. G-CSF-mobilized NK cell TNF-α and IFN-γ secretion was impaired at day 0 compared to healthy donors but was progressively restored after culture. In conclusion, expansion of NK cells from G-CSF-mobilized sources may progressively improve their functionality.