Activated B cells and T cells express CD154/CD40 ligand in vitro. The in vivo expression and function of B cell CD154 remain unclear and therefore were examined. Tonsillar B and T cells expressed CD154 at a similar density both in situ and immediately ex vivo, whereas a significantly higher percentage of the former expressed CD154. CD154-expressing B cells were most frequent in the CD38 positive IgD+ pre-germinal center (GC)/GC founder, CD38 positive GC and CD38− IgD− memory populations, and were also found in the CD38− IgD+ naive and CD38 bright IgD+ plasmablast subsets, but not in the CD38 bright IgD− plasma cell subset. B cell expression of CD154 was induced by engaging surface Ig or CD40 by signals that predominantly involved activation of AP-1/NF-AT and NF-κB, respectively. The functional importance of CD154-mediated homotypic B cell interactions in vivo was indicated by the finding that mAb to CD154 inhibited differentiation of CD38 positive IgD− GC B cells to CD38− IgD− memory cells. In addition, mAb to CD154 inhibited proliferation induced by engaging sIg or CD40, indicating the role of up-regulation of this molecule in facilitating B cell responsiveness. Of note, CD154 itself not only functioned as a ligand but also as a direct signaling molecule as anti-CD154-conjugated Sepharose beads costimulated B cell responses induced by engaging surface Ig. These results indicate that CD154 is expressed by human B cells in vivo and plays an important role in mediating B cell responses.