Chimeric antigen receptor (CAR)-T cell therapy possesses the potential to cause unexpected on-target toxicities that may be life-threatening. Non-human primates (NHPs) share considerable structural homology and expression profiles of most proteins with humans and are therefore utilized as an animal model for non-clinical safety studies of CAR-T cells. We have developed a lymphodepleted NHP model by conditioning the animals with immunosuppressive chemotherapy designed to simulate clinical practice conditions, to induce transient mixed chimerism before the administration of human EPHB4 receptor-specific CAR-T cells to evaluate the toxicity of these cells. Lymphodepletion was successfully achieved on day -1 before T cell infusion and persisted over 7 days without severe organ toxicities. A single administration of human EPHB4-CAR-T cells did not induce overt on-target/off-tumor toxicities during 7 days after the administration, although CAR-T cells were activated in vivo as evidenced by the elevation in copy numbers of the CAR transgene. Although this NHP model is limited for the full evaluation of toxicity of human CAR-T cells and the conditioning protocol should be further optimized, this lymphodepleted NHP model could be used to assess acute on-target/off-tumor toxicities of CAR-T cells.