[Abstract]  Objective: The aim of this study was to express CD40L, IL-2, IL-4, IL-21 and BAFF in NIH3T3 cells to further optimize the in vitro culture conditions of B cells. Methods: Lentiviral expression vectors containing CD40L, IL-2, IL-4, IL-21, and BAFF genes were constructed by molecular cloning, lentiviral infection of NIH3T3 cells was prepared, and the expression of CD40L and BAFF was detected by flow cytometry, the secretion levels of IL-2, IL-4, and IL-21 were detected by ELISA, and the lentiviral expression vector was used as a feeder cell and B cells were in co-cultured in vitro for 16 days, the proliferation of B cells was observed microscopically, and the secretion level of IgG was detected by ELISA. Results: In this study, we successfully constructed the NIH3T3-CD40L-IL-2-IL-4-IL-21-BAFF cell line, which was able to stably express CD40L, BAFF, and secrete high levels of IL-2, IL-4, and IL-21, and co-cultured with B cells in vitro for 16 days was able to support the efficient expansion of low-density B cells and induce the differentiation of B cells into plasma cells, and its antibody secretion was as high as 517 ng/mL.Conclusion: In this study, we successfully constructed feeder cell lines capable of supporting B cell proliferation and activation in vitro, which laid a good foundation for the preparation of monoclonal antibodies.