Natural killer (NK) cells have an important function in the anti-tumor response early after stem cell transplantation (SCT). As part of a prospective randomized phase III study, directly comparing the use of CD3+/CD19+-depleted peripheral blood stem cell (PBSC) harvests with CD34+-selected PBSC harvests in allogeneic human leukocyte antigen-matched SCT, we here show that the use of CD3+/CD19+-depleted PBSC grafts leads to early NK cell repopulation and reconstitution of the CD56dim and CD56bright NK cell subsets, with concomitant high cytolytic capacity. In the CD34 group, this process took significantly longer. Moreover, in the CD3/19 group after reconstitution, a higher percentage of killer immunoglobulin-like receptor-positive NK cells was found. Although similar percentages of CD94-positive NK cells were found in both groups, in the CD34 group, almost all expressed the inhibitory CD94:NKG2A complex, whereas in the CD3/19 group, the inhibitory CD94:NKG2A and the activating CD94:NKG2C complex were equally distributed. This preferential development of NKG2C-expressing NK cells in the CD3/19 group was paralleled by a loss of NKG2A-mediated inhibition of NK cell degranulation. These results show that the use of CD3+/CD19+-depleted grafts facilitates strong NK cell cytolytic responses directly after SCT, and the rapid emergence of an NK cell receptor phenotype that is more prone to activation.