Human cytomegalovirus (CMV)-specific cytotoxic T lymphocyte (CTL) immune response must be reconstituted for long-term protection against CMV relapse and disease in hematopoietic stem cell transplantation (HSCT) recipients. We phenotypically quantitated absolute numbers of CMV-pp65 peptide-specific CTLs (CTL(CMV)) in 50 related donor unmanipulated allografts infused into HLA-matched or -mismatched recipients and examined the incidence of CMV reactivation. High CTL(CMV) with terminally differentiated effector CD45RO(-)CD62L(-) cell (T(EMRA)) phenotype in the allografts were associated with reduced risk of CMV reactivation, in the presence of sufficient CD45RO(+)CD62L(-) cell (T(EM)) infusion (>/=0.208 x 10(6)/kg). Early after transplantation, there was significant expansion of CTL(CMV) with the central memory CD45RO(+)CD62L(+) cell (T(CM)) phenotype when CMV was reactivated. The frequencies of CTL(CMV) T(Naive) (CD45RO(-)CD62L(+)), T(CM), and T(EM) at day 90 posttransplantation and of CTL(CMV) T(EMRA) at day 60 posttransplantation were greater in recipients with higher infusions of CTL(CMV) T(EMRA), suggesting protective immunity transferred by infusion of CTL(CMV) within allografts. Moreover, the majority of the CTL(CMV) identified in the recipients early after HSCT was of donor origin. Our findings support that measuring levels of CTL(CMV) and its subsets in the donor grafts and manipulating these cells early after transplantation may help control CMV reactivation, which is closely correlated with immune reconstitution and differentiation of CTL(CMV) subsets.