Introduction: Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) infection remains one of the most important complications that potentially causes non-relapse mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Plasmacytoid dendritic cells (pDC) are a sentinel cell type with potent capacity of recognizing viral antigens and producing massive type I interferon, which contributes to the effective elimination of viral infection. The association of pDC in allogeneic grafts (allo-grafts) with CMV and EBV infection is widely unknown. Here we aim to investigate the association of pDC dose infused with CMV and EBV infection within 100 days after HSCT. Methods: A total of 100 patients receiving allo-HSCT at the Children's Hospital of Soochow University were included. A cohort of 80 patients was enrolled from September 2020 to June 2021 (discovery cohort); another cohort of 8 patients was enrolled from July 202 to August 2022 (validation cohort). Conditioning regimens based on busulfan/cyclophosphamide (Bu/Cy) included myeloablative, reduced intensity and non-myeloablative conditioning. Copies of viral genomes in peripheral blood were detected weekly after HSCT using real-time quantitative polymerase chain reaction. Patients who tested positive for CMV viremia were given ganciclovir as preemptive therapy, while patients with EBV viremia were treated with upfront rituximab. Samples of allogeneic grafts (allo-grafts) from corresponding donors were collected to examine DC subsets and T-cell subsets by flow cytometry. The discovery cohort was analyzed to investigate the association of pDC dose infused with CMV and EBV infection within 100 days after HSCT; the validation cohort was analyzed to verify this association and investigate its correlation with pDC reconstitution by testing patients’ circulating pDC concentration on day +30 after HSCT. Results: At baseline, mean age of discovery cohort was 8 years (range, 1-15 years); the diagnosis included acute myeloid leukemia (AML) (n=22, 28%), myelodysplastic syndrome (MDS) (n=4, 5%), acute lymphoblastic leukemia (ALL) (n=24, 30%), aplastic anemia (n=20, 25%), and other diagnosis (n=10, 12%) requiring HSCT. Within 100 days after HSCT, 54 patients (68%) developed CMV viremia at a median of 28.5 days (range, 13-65 days); multivariate analysis suggested that the high pDC dose infused was an independent protective factor for CMV viremia (HR, 0.456; 95% CI, 0.247-0.843; p = 0.012). Within 100 days after HSCT, 41 patients (51%) developed EBV viremia at a median of 24 days (range, 11-90); multivariate analysis suggested that the high dose of pDC infused was an independent protective factor for EBV viremia (HR, 0.499; 95% CI, 0.257-0.967; p = 0.040). In the validation cohort, patients developing CMV and EBV viremia within 100 days after HSCT had significantly lower pDC dose in graft compared to patients without CMV and EBV viremia. On day +30 after HSCT, circulating pDC concentration was higher in patients without CMV and EBV viremia than those who developed viremia. Additionally, patients with a higher pDC dose infused had a correlation with a higher circulating pDC concentration on day +30 after HSCT (r = 0.68). Conclusions: In this retrospective study, we observed that pDC dose infused might be a protective factor for CMV and EBV infection early after HSCT. In addition to the inherent ability to produce antiviral immune responses, a high pDC dose may lower the risk of viral infection by enhancing pDC reconstitution early after HSCT.