<h3>Purpose/Objective(s)</h3> Radiation-induced lymphopenia is well documented in craniospinal irradiation (CSI), and recent work suggests proton CSI (pCSI) decreases hematotoxicity associated with the technique. However, no studies have examined the relative contribution of CSI subvolumes on lymphopenia rates. The objective of this study was to evaluate the impact of brain, spine, and boost target volume (bPTV) dosimetry on acute lymphopenia in pediatric patients receiving pCSI. <h3>Materials/Methods</h3> This single-institution, retrospective study evaluated patients aged ≤25 receiving pCSI ± sequential boost as part of definitive therapy for CNS tumors. The absolute lymphocyte count (ALC) at nadir during RT was recorded. Baseline differences in patients stratified by grade of lymphopenia were tested with Mann-Whitney U and chi-square tests. Difference in median overall survival (OS) was evaluated using the log-rank test. Simple and multiple regression was used to identify dosimetric quantities associated with ALC employing stepwise variable selection with significance set at p ≤ 0.05. <h3>Results</h3> From 5/2014 - 1/2020, 53 patients were treated with pCSI with a median follow up of 4.5 years. Tumors included medulloblastoma (66%), dysgerminoma (17%), pineoblastoma (8%), ependymoma (4%), ATRT (2%), neuroblastoma (2%), and PNET (2%). Of these, 28%, 66%, and 74% received neoadjuvant, concurrent, and adjuvant chemotherapy, respectively. Grade 1, 2, 3, and 4 lymphopenia during RT was observed in 6%, 6%, 59%, and 30%, respectively. There was no significant difference in sex, age, craniospinal axis dose, boost dose, bPTV, performance status, pretreatment ALC, vertebral body sparing (VBS), or concurrent chemotherapy in those with and without G3/G4 lymphopenia. However, there was a significantly lower proportion of patients receiving neoadjuvant chemotherapy in the G3/G4 lymphopenia group (23% vs 67%, p=0.047). There was a nonsignificant trend for poorer OS in those experiencing G3/G4 lymphopenia during RT (median OS: 3.8 yrs vs 4.7 yrs). On simple regression, metrics describing dose to the brain, spine, and bPTV were significantly associated with ALC (<b>Table 1</b>). On multiple regression controlling for age and neoadjuvant chemotherapy, ALC was significantly associated with mean doses to the spine and bPTV. <h3>Conclusion</h3> Higher mean doses to the spine and bPTV were associated with lower ALC during pCSI in this pediatric population. This effect remained when controlling for age and neoadjuvant chemotherapy, factors known to be associated with lymphopenia during therapy. Larger studies are necessary to further elucidate the impact of pCSI subvolumes, particularly marrow-containing structures within the spine and pelvis, on acute lymphopenia rates in pCSI.