Although traditionally associated with IgE-mediated atopy, mast cells can also regulate adaptive immune responses. We have developed a novel co-culture system using IL-4-treated bone marrow-derived mast cells to activate a distinct subset of B cells in vitro. Using transwells to separate the cells showed that contact was not required and that the phenotype of these cells closely resembled the regulatory B cells (BRegs) we have identified in vivo. Indeed, when these mast cell-activated B cells were adoptively transferred in vivo, they suppressed the induction of cell-mediated immunity. Significant increases in intracellular IL-10 and IL-13, as well as both subunits of IL-35 (EBI3 and p35) strongly suggests a role for these immunosuppressive cytokines. We have also discovered that mast cell-derived microparticles are responsible for activating B cells. Using these microparticles rather than the IL-4-treated mast cells themselves was more than sufficient to activate B cells that were immune regulatory in vivo. Revelation of the consequences of mast cell-B cell interactions together with the mechanisms involved will allow us to manipulate BReg activation in vivo. This is likely to be important for a range of disorders including protecting from autoimmune disease and combatting cancer.