Chimeric antigen receptor (CAR) T cell therapy is a powerful adoptive immunotherapy against blood cancers, but the therapeutic effect was not efficient enough on solid tumors. B cells have been reported to play a critical role in regulating memory T differentiation and cytotoxic T development. However, as of yet the influence of such B cells on CAR T cells has not been discussed. In this study, using ephrin type-A receptor 2 (EphA2) specific CAR T cells, we cultured B cells successfully to stimulate T cells in vitro, and investigated the cell differentiation and anti-tumor efficiency. We observed that EphA2-CAR T cells stimulated by B cells performed enhanced anti-tumor ability with more interferon γ (IFN γ) production and higher OX40 expression. The differentiation of CAR T cells was arrested after B cells stimulation for more than 7 days with the percentage of central memory T cells (Tcm) increasing. In addition, next generation sequencing was performed. The top expressed genes clustered in activation, leukocyte migration and chemokine signaling pathway contributed to the anti-glioblastoma (GBM) activity of CAR T cells stimulated by B cell. In conclusion, these results indicated the importance of B cells in retarding CAR T cells differentiation and enhancing anti-tumor activity, which paves the way for the rapid exploitation of EphA2-CAR T cells against GBM in the future. Overall design: EphA2-CAR T cells stimulated with or without B cells were co-cultured with U251 or U373 (1:1). The suspended cells were collected and washed with PBS for three time. CD3+ T cells were isolated using Dynabeads™ CD3 (Invitrogen). Total RNA was extracted from cells attached on the beads using TRIzol Reagent (Invitrogen). The culture dish was washed with PBS for three times and RNAs of adherent cells were collected using TRIzol reagents. Samples containing 2 µg total RNA each were used as input material for generating the sequencing libraries by company.