Background: Chimeric antigen receptor (CAR) T-cells are highly effective for patients with relapsed or refractory B-ALL. Lymphodepletion has been paramount to CAR T-cell expansion and efficacy. Recent data identified an association of fludarabine exposure with reduced relapse rates, longer leukemia free survival, and more prolonged B-cell aplasia following tisagenlecleucel. However, the relationship of fludarabine exposure to lymphopenia and the mechanism by which dosing impacts outcomes following CAR T-cells remains unknown. Methods: We conducted a retrospective review of children and young adults with B-ALL who received fludarabine for lymphodepletion (LD) prior to infusion of anti-CD19, anti-CD22, or anti-CD19/22 bispecific CAR T-cells. The primary objective of this study was to investigate the impact of fludarabine on the depth and duration of lymphopenia. Secondary objectives included examining the impact of fludarabine exposure on: 1) overall clinical response, and 2) CAR T-cell expansion. All patients in the analysis cohort received fludarabine 25 mg/m²/day x 3 days ( = 75 mg/m² cumulative dosing). Fludarabine exposure (AUC per dose) for each patient was predicted using a validated population PK model and analyzed with various responses/endpoints using the 3-day cumulative exposure/patient. Results: Of 137 children and young adults, the median cumulative AUC was 8.3 mg*hr/mL (range, 3.00-13.83 mg*hr /mL). The overall complete response (CR) rate was: 67.9% (93/137). The median duration of lymphopenia (defined as an absolute lymphocyte count (ALC) £ 150 K/uL) was 5 days (range, 0-15); and the median ALC on Day 0 was 80 K/uL (range, 0-1250 K/uL). When stratified by the median fludarabine exposure, there was no difference in depth or duration of lymphopenia or CAR T-cell expansion collectively or per trial. The median fludarabine exposure also did not differ between responders and non-responders. However, depth and duration of lymphopenia did differ in the CD22 CAR T-cell trial. Specifically, in those achieving CR, the duration of lymphopenia was longer than in non-responders (7 days versus 4 days, p = 0.030). Similarly, CAR T-cell expansion was higher if lymphopenia duration was greater than 5 days (p = 0.019). These findings were not seen across our less persistent CD19 or CD19/22 CAR T-cell trials. Conclusions: Although lymphodepletion impacts CAR T-cell response, we did not find an association between CR rate, depth of lymphopenia, or duration of lymphopenia with varying fludarabine exposures. This raises additional questions regarding the impact of LD therapy on CAR T-cell outcomes—especially since a more prolonged lymphopenia was associated with high CR rates and greater peak expansion in at least one of our studies (CD22 CAR T-cells). Future analysis will evaluate the impact of fludarabine on Day 0 cytokines and other immune parameters. Clinical trial information: NCT03448393.