CD40, a glycoprotein expressed on B lymphocytes plays an important role in B cell development, growth and differentiation. The ligand for the CD40 is a 39‐kDa glycoprotein (CD154) expressed onthe surface of activated T lymphocytes and is essential for thymus‐dependent humoral immunity. The expression of CD154 is tightly regulated and its transient expression reduces the chances of potentially deleterious bystander activation of B cells. Stimulation through CD40 has been studied in vitro by using antibodies against CD40, by membranes of activated T cells or lately, by CD154 transfected cells. In this work we have evaluated the outcome of CD40‐CD40 ligand interaction in vitro and in vivo by using CD154‐transfected L929 cells. In vitro assaysshowed that CD154‐L929 cells can induce on B cells: IL‐4‐dependent proliferation, up‐regulation of CD23, CD54 and class II molecules and can also rescue WEHI‐231 B cell lymphoma from anti‐IgM‐induced apoptosis. Interestingly, in vivo assays revealed that when CD154‐L929 cells were inoculated into the spleen, mice developed a strong but transient production of anti‐erythrocyte autoantibodies. Through B lymphocyte activation with CD154‐transfected L929 cells both in vitro and in vivo, our data reveal that enforced and prolonged expression of CD40 ligand overcomes the tightly regulated mechanisms of B cell activation, triggering the production of autoantibodies. This system might be used to evaluate the early steps of an autoimmune response and the role of CD40‐CD154 in the induction of primary responses in vivo.