Abstract Reactivation of latent cytomegalovirus (CMV) is increased in recipients of allogeneic hematopoietic cell transplantation (allo-HCT) with seropositive CMV using posttransplant cyclophosphamide (PT-Cy)–based graft-versus-host disease (GVHD) prophylaxis. Letermovir, a novel DNA terminase complex inhibitor, reduces the incidence of clinically significant CMV infection (csCMVi) in this population; however, parameters that predict csCMVi after letermovir withdrawal are not well described. Here, we examined clinical and immunological parameters in 294 recipients of PT-Cy–based allo-HCT, including 157 patients with CMV, of whom 80 completed letermovir prophylaxis without csCMVi and subsequently stopped letermovir. In this population, the median duration of letermovir exposure was 203 days (interquartile range [IQR], 160-250 days). After letermovir withdrawal, the 90-day cumulative incidence of csCMVi was 23.0% (95% confidence interval, 14.3-32.8). There were no episodes of CMV end-organ disease. Hypogammaglobulinemia before letermovir discontinuation was predictive of csCMVi (hazard ratio, 0.33; 95% confidence interval, 0.12-0.93; P = .03), whereas T-cell and B-cell reconstitution before letermovir withdrawal were not predictive of csCMVi. Higher numbers of natural killer cells were found before letermovir withdrawal in patients who experienced csCMVi (median, 202 vs 160; P = .03). In recipients with seropositive CMV, CD3+CD4–CD8+ T-cell reconstitution was faster in patients with CMV regardless of letermovir exposure. Taken together, these data suggest that csCMVi after letermovir withdrawal was frequent in patients treated with PT-Cy, despite prolonged exposure. Strategies to boost CMV-specific adaptive immunity in patients with persistent hypogammaglobulinemia is a logical pathway to reduce csCMVi after letermovir withdrawal.