Mass cytometry was used to investigate the effect of CMV reactivation on lymphocyte reconstitution in hematopoietic cell transplant patients. For eight transplant recipients (four CMV negative and four CMV positive), we studied PBMCs obtained 6 mo after unrelated donor hematopoietic cell transplantation (HCT). Forty cell-surface markers, distinguishing all major leukocyte populations in PBMC, were analyzed with mass cytometry. This group included 34 NK cell markers. Compared with healthy controls, transplant recipients had higher HLA-C expression on CD56- CD16+ NK cells, B cells, CD33bright myeloid cells, and CD4CD8 T cells. The increase in HLA-C expression was greater for CMV-positive HCT recipients than for CMV negative recipients. Present in CMV-positive HCT recipients, but not in CMV-negative HCT recipients or controls, is a population of killer cell Ig-like receptor (KIR)-expressing CD8 T cells not previously described. These CD8 T cells coexpress CD56, CD57, and NKG2C. The HCT recipients also have a population of CD57+NKG2A+ NK cells that preferentially express KIR2DL1. An inverse correlation was observed between the frequencies of CD57+NKG2C+ NK cells and CD57+NKG2A+ NK cells. Although CD57+NKG2A+ NK cells are less abundant in CMV-positive recipients, their phenotype is of a more activated cell than the CD57+NKG2A+ NK cells of controls and CMV-negative HCT recipients. These data demonstrate that HCT and CMV reactivation are associated with an increased expression of HLA-C. This could influence NK cell education during lymphocyte reconstitution. The increased inhibitory KIR expression by proliferating CMV-specific CD8 T cells suggests regulatory interactions between HLA-C and KIR might promote Graft-versus-Leukemia effects following transplantation.