Abstract Preclinical in vivo models are used to profile or refine CAR T therapies before advancing to human studies. Establishing long-term CAR T persistence and efficacy continues to challenge progress in the CAR T space, thus development of robust platforms that can provide longitudinal assessments of CAR T persistence and functionality is paramount. To this end, we used the NALM6-Luc ALL model to develop a flow cytometry platform that provides quantitative analysis of CAR T cells over time as well as surface markers that are documented to correlate with sustained T cell persistence and activation in vivo. Tumored NSG mice were enrolled into treatment groups based on tumor burden calculated from bioluminescence imaging (BLI) data. T cells transduced to express a CD19 CAR (or left untransduced (UTD)) were injected intravenously at 1.0E+07 cells/mouse. Tumor burden was monitored by BLI and flow cytometry was performed weekly on survival bleeds to measure CAR T persistence and examine phenotype. Treatment with CD19 CAR T cells delayed tumor growth resulting in an increase in time to progression of 107.1% compared to UTD controls. However, no regressions were observed. On day 1 post-transfer, CD3+ T cells were detectable in mice that received both CAR T and UTD cells (140+/-26 and 162+/-56 cells/uL blood respectively). T cells declined to near undetectable levels by Day 20, a point when all animals in the UTD treatment group had reached euthanasia criteria. After Day 20, T cells expanded in circulation in the CD19 CAR T treatment group reaching 695+/-327 cells/uL blood by day 40. T cell expansion coincided with exponential tumor outgrowth in the treatment group. To assess T cell functionality, flow cytometry was used to measure the expression of biomarkers for T cell activation (CD25, 4-1BB, and ICOS) and exhaustion markers (TIM-3, PD-1, and LAG-3). CD25, 4-1BB, and ICOS expression did not exceed positivity on more than 15% of CD8+ T cells and peaked by day 30 before downregulation was observed. Notably, PD-1 and LAG-3 expression levels continued to increase throughout the study, suggesting T cells were taking on an exhausted phenotype. Similar trends were observed on CD4+ T cells. To investigate whether the late phase T cell expansion was a graft vs. host response, CAR T cell measurements were compared to non-tumored animals. Expansion as well as PD-1/LAG-3 expression was only observed in tumor-bearing mice indicating the responses were tumor-specific. Taken together, these data demonstrate that CD19 CAR T cells can inhibit NALM6-Luc tumor growth in vivo and expand in circulation in an antigen-specific manner. Furthermore, CAR T cell failure to control tumor growth may be due to onset of an exhausted phenotype. Finally, we demonstrate that flow cytometry can be used to characterize T cell persistence and functionality in murine xenograft tumor models. Citation Format: David W. Draper, Derrik Germain, Stacey Roys, Olivia Nelson, Scott Wise. Preclinical assessment of chimeric antigen receptor (CAR) T persistence and functionality in the disseminated NALM6-Luc human B cell acute lymphoblastic leukemia (ALL) model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2749.