Purpose : CMV-specific cell-mediated immunity (CMI) is a well-known predictor for the increased risks of CMV infection in patients received allo-HSCT. We tried to evaluate the clinical usefulness of CMV immune monitoring by ELISPOT assay which was relatively easy to standardize to implement the T cell immune monitoring in a clinical setting of pediatric allo-HSCT Methods : From March 2016 through February 2017, children ≤18 years of age receiving allogeneic HSCT at Asan Medical Center were prospectively enrolled. All subjects underwent CMV pp65 and IE1-specific ELISPOT assays before transplantation and then monthly until detection of CMV-specific T cells by ＞ 5 spots/2.0 x 105 cells. We analyzed the risk factor for the clinically significant CMV burden including prolonged or recurrent or late onset CMV infection and tissue-invasive CMV disease. Results : A total of 40 recipients with 23 haploidentical donor, 8 sibling donor and 9 unrelated donor HSCT were enrolled. The pre-transplant CMV status was D+/R+ in 85.0%, D-/R+ in 7.5% and D+/R- in 5.0%. CMV reactivation was observed in 27 patients including 3 with CMV retinitis and median time to initial CMV reactivation was 40 days (range, 25-69 days). The presence of CMV specific CMI before HSCT and transplantation for other causes than hematologic malignancies were significant factors for CMV infection after HSCT (adjusted odds ratio [aOR] 29.9; P= 0.047 and aOR 136.6; P = 0.025, respectively). Overall, 33 patients recovered CMV-specific immunity at a median of 34 days post SCT (range, 29 – 81 days) and 75.0% of recipients showed CMV-specific ELISPOT response at 60 days. Recipients with no ELIPSOT response during the follow-up period were younger than those with positive response (1.9 vs. 9.3 years of age; P=0.005). Prolonged, recurrent, and late-onset CMV infection was observed in 18, 1, and 3 recipients, respectively. No patients died from cytomegalovirus infection. The presence of CMV specific CMI before HSCT had the most significant influence on the clinically significant CMV burden (aOR 8.9, P=0.021). Conclusions : Among pediatric allo-HSCT recipients, significant burden of CMV infection was associated with absence of CMV ELISPOT response before HSCT. The usefulness of the CMV ELISPOT assay along with viral load monitoring should be confirmed in a large-scale trial.