There has been a growing interest in the use of B cells for cancer vaccines, since they have yielded promising results in preclinical animal models. Contrary to dendritic cells (DCs), we know little about the migration behavior of B cells in vivo. Therefore, we investigated the interactions between CD40-activated B (CD40B) cells and cytotoxic T cells in vitro and the migration behavior of CD40B cells in vivo. Dynamic interactions of human antigen-presenting cells (APCs) and T cells were observed by time-lapse video microscopy. The migratory and chemoattractant potential of CD40B cells was analyzed in vitro and in vivo using flow cytometry, standard transwell migration assays, and imaging of fluorescently labeled murine CD40B cells. Murine CD40B cells show migratory features similar to human CD40B cells. They express important lymph node homing receptors which were functional and induced chemotaxis of T cells in vitro. Striking differences were observed with regard to interactions of human APCs with T cells. CD40B cells differ from DCs by displaying a rapid migratory pattern undergoing highly dynamic, short-lived and sequential interactions with T cells. In vivo, CD40B cells are home to the secondary lymphoid organs where they accumulate in the B cell zone before traveling to the B/T cell boundary. Moreover, intravenous (i.v.) administration of murine CD40B cells induced an antigen-specific cytotoxic T cell response. Taken together, this data show that CD40B cells home secondary lymphoid organs where they physically interact with T cells to induce antigen-specific T cell responses, thus underscoring their potential as cellular adjuvant for cancer immunotherapy.