We examined cytomegalovirus-specific cell-mediated immunity (CMV-CMI) to pp65 and IE-1 at 100 days after hematopoietic cell transplantation (HCT) following discontinuation of letermovir prophylaxis using ELISPOT (T-SPOT®.CMV, Oxford-Immunotec, Abingdon, UK). We compared ELISPOT results to a laboratory-developed intracellular cytokine staining (ICS) assay and characterized thresholds for the prediction of late clinically significant (cs)CMV infection using receiver operating characteristic analysis. We identified factors associated with high (i.e., at or above threshold) CMV-CMI to both antigens. ELISPOT correlated well with polyfunctional CMV-specific T-cell immunity by ICS. We defined thresholds of 67 and 4 spots per 250,000 cells for pp65 and IE-1, respectively, for predicting late csCMV infection. PBSC graft source, CMV seropositive donor, and/or CMV reactivation in the first 100 days post-HCT were associated with high CMV-CMI to pp65 and/or IE-1. Patients with high CMV-CMI to both antigens at day 100 had a lower incidence of late csCMV infection, however, late changes in immunosuppression affected risk prediction. Clinical risk factors (e.g., early CMV infection, acute graft-versus-host disease) predicted late csCMV and improved the predictive value of CMV-CMI testing. Thus, standardized CMV-CMI, after HCT, combined with clinical factors can be used to accurately stratify the risk of late csCMV infection after letermovir prophylaxis.