A large number of infectious diseases caused by viral or bacterial infections are treatable and/or preventable by vaccination. In addition, ongoing research is aimed at the development of vaccines against other types of diseases, including almost all forms of cancer. The efficacy of a vaccine relies on the antigen-specific response by the entire repertoire of immune competent cells. Here, we have generated a powerful mitogen fusion protein, CD40L-FasL-IgFc, which stimulates CD40(+) cells robustly. We found that this specific cell activation is accompanied by increased expression of PRDI-BF1 (Blim-1) RNA, an indicator of terminal B-cell differentiation, in cultures stimulated with CD40L-FasL-IgFc. The addition of specific inhibitors of NF-kappaB and MEK1/2 partially suppressed the observed proliferative effects of CD40L-FasL-IgFc. When tested in vivo, the immune response to influenza HA vaccine was significantly increased by co-administration of CD40L-FasL-IgFc. Moreover, the co-administration of the cDNA expression plasmid encoding CD40L-FasL-IgFc significantly boosted the vaccine response. We now have a unique opportunity to evaluate our novel fusion protein adjuvant, and other similarly constructed fusion proteins, in both protein-based and genetic vaccines.