Transitional immature B cells undergo apoptosis and fail to proliferate in response to BCR cross-linking, thus representing a target for negative selection of potentially autoreactive B cells in vivo. In agreement with recent reports, transitional B cells were divided into developmentally contiguous subsets based on their surface expression of CD23. When transferred, CD23(+) transitional B cells readily localized to the splenic follicles and the outer PALS. Compared with CD23(-) transitional B cells, CD23(+) transitional B cells proliferated more vigorously and were rescued from BCR-induced apoptosis to a greater degree, by T cell help signals. However, both CD23(-) and CD23(+) transitional B cells failed to up-regulate CD86 (B7-2) in response to BCR ligation. These findings demonstrate that phenotypically defined subsets within the transitional B cell population are functionally distinct. Specifically, responsiveness to T cell help is a late acquisition corresponding to the stage when the B cells gain access to peripheral compartments enriched in antigen and activated T cells. The failure of transitional B cells to up-regulate CD86 to BCR-mediated stimulation suggests a unique interaction between transitional B cells and T cells with implications for tolerance in the T cell compartment.