STAT6 deficient Treg-of-B (P) cells expressed lower LAG3 level and suppressive ability. In order to demonstrate the important role of STAT6 for Treg-of-B (P) cell generation, genetic deletion of STAT6 or the chemical inhibition of STAT6 phosphorylation were carried out. Wild type (WT) and STAT6 knockout (STAT6KO) CD25- CD4+ T cells were cultured with Peyer’s patch B cells or wild type CD25-CD4+ T cell cultured with Peyer’s patch B cell in presence of DMSO (control group) or AS (STAT6 inhibition group) for 3 days. After three-day cultured, Treg-of-B (P) cells were harvested and applied for FACS analysis, including phosphorylated STAT1, 3, 5, and 6 (A), LAG3 expression (B) and suppressive function test (C). (D) The cytokine levels of IL-4, IL-10, and IFNγ secreted by WT and STAT6KO Treg-of-B (P) cells. After 3-day cocultured, Peyer’s patch B cells were depleted, Treg-of-B (P) cells were harvested and re-stimulated with plate-bound anti-CD3 plus soluble CD28 1 µg/ml. after 48 h stimulation, the supernatant was collected for cytokine evaluation by ELISA. Results are expressed as the mean ± SEM. Data are representative of three to five different experiments. **p < 0.01, ***p < 0.005 compared with the wild type Treg-of-B (P) cells or responder T only group. #p < 0.05, ###p < 0.005, compared with wild type or DMSO treated Treg-of-B (P).