For a long time, it has been thinking that B cells regulate immune responses by producing antigen-specific antibodies. However, previous studies have revealed that specific B-cell subsets can also negatively regulate T-cell immune responses, and have been termed regulatory B cells. Recently, our study showed that mouse CD1d(hi)CD5(+) B cell subsets mainly produce IL-10. Therefore, we named these populations B10 cells. In our previous studies have also indicated that human B10 cells with the ability to express the inhibitory cytokine interleukin (IL)-10 have been identified. Although it is rare, B10 cells are potent negative regulators of antigen-specific inflammation and T-cell-dependent autoimmune diseases in mice. How B10-cell IL-10 production and regulation of antigen-specific immune responses are controlled in vivo without inducing systemic immunosuppression is unknown. Using an experimental autoimmune encephalomyelitis, which is a mouse model for multiple sclerosis, we have shown that B10-cell maturation into functional IL-10-secreting effector cells that inhibit in vivo autoimmune disease requires IL-21 and CD40-dependent cognate interactions with T cells. In addition, the ex vivo provision of CD40 and IL-21 receptor signals can drive B10-cell development and expansion by four-million-fold, and generate B10 effector cells producing IL-10 that markedly inhibit disease symptoms when transferred into mice with established autoimmune disease. The ex vivo expansion and reinfusion of autologous B10 cells may provide a novel and effective in vivo treatment for severe autoimmune diseases that are resistant to current therapies.