The incorporation of anti-thymocyte globulin (ATG) into conditioning regimens to prevent graft-versus-host disease (GVHD) can markedly impair immune reconstitution (IR). We examined the associations among ATG exposure, recipient lymphocyte counts, IR, and post-transplant outcomes. This retrospective study included patients aged ≤18 years who underwent allogeneic hematopoietic stem cell transplantation (HSCT) from April 2005 to April 2020. Outcomes assessed were GVHD incidence, overall survival (OS), and IR, evaluated via thymic magnetic resonance imaging (MRI) and quantification of CD4+ T cells and recent thymic emigrants (RTEs). Patients receiving ATG were stratified into low (ATG/lymphocyte ratio <0.01) and high (ATG/lymphocyte ratio >0.01) subgroups. The low-ratio group experienced a higher GVHD incidence (29 [59%] vs. 7 [16.6%]) but demonstrated superior IR in both laboratory and MRI assessments (p < 0.0001). Median thymic volume was significantly greater in the low-ratio group (14.7 cm³ vs. 4.5 cm³, p < 0.001), correlating with improved OS and reduced transplant-related mortality (TRM) (80.4% vs. 58.0%, p = 0.031; 13.1% vs. 33.0%, p = 0.035). Tailoring ATG dosing to individual patients may enhance thymic recovery and optimize transplant outcomes.