Peyer's patches, splenic, and peritoneal B cells induce regulatory T (Treg-of-B) cells in vitro without exogenous chemicals and cytokines. Treg-of-B cells exert suppressive function both in vitro and in vivo. Here, we demonstrate experimental procedures for the generation and characterization of Treg-of-B cells. We describe steps for isolating B220 B cells, isolating CD4CD25 T cells, inducing Treg-of-B cells, identifying Treg-of-B cells by flow cytometry, cytokine analyzing by ELISA, and functional testing by suppression assay. For complete details on the use and execution of this protocol, please refer to Chien et al. and Chu et al..