The antitumor activity of natural killer (NK) cells has recently been shown to be assessable at a single-cell level via flow cytometric detection of CD107a. We used this novel method to prospectively quantify and characterize tumor-reactive NK cells in patients undergoing myeloablative or nonmyeloablative conditioning and allogeneic hematopoietic stem cell transplantation (HSCT). Degranulation of NK cells in the peripheral blood of 34 patients after HSCT (day +30, day +90) was determined by evaluating CD107a expression after coincubation of the cells with tumor targets. The percentage of degranulating NK cells was higher after nonmyeloablative conditioning than after myeloablative conditioning (P<.001), indicating a higher activation state and increased antitumor activity of NK cells after nonmyeloablative conditioning. We were able to analyze NK cell subsets separately and found that CD56bright NK cells following HSCT are functionally different from CD56bright NK cells in healthy donors, as indicated by a high percentage of degranulating NK cells in response to tumor targets in patients after HSCT. The CD107a assay is a new and feasible method to quantify and characterize tumor-reactive NK cells after HSCT. Using this method, we found that NK cells had high antitumor cytotoxic activity after nonmyeloablative conditioning, which may contribute to the effectiveness of nonmyeloablative conditioning.