Although a positive cytotoxic crossmatch (XM) has been reported to predict graft failure, mainly in solid organ transplantations, its significance in allogeneic hematopoietic cell transplantation (HCT) remains to be elucidated. We retrospectively assessed the impact of positive XM on neutrophil engraftment in 41 patients who underwent HCT with an HLA-mismatched related donor. XM was positive in 22 patients. Six of these 22 patients were also positive for anti-HLA antibody, whereas only 1 was positive for donor-specific anti-HLA antibody. The cumulative incidence of engraftment at day +28 was 89.5% in patients with negative XM versus 59.1% in those with positive XM (P = .08). In particular, positive B cell warm XM was significantly associated with a lower probability of engraftment at day +28 (46.7% versus 88.5%; P = .04). In a multivariate analysis, both positive XM and positive B cell warm XM were significantly associated with delayed engraftment (hazard ratio [HR], .46; P = .02 and HR, .41; P = .01, respectively). There was no significant difference in the achievement of engraftment between those with and without detection of anti-HLA antibodies. In conclusion, positive XM might be associated with a delayed neutrophil engraftment after HCT from HLA-mismatched related donors.