The interactions of CD28‐B7 and CD40‐CD40 ligand (CD40L) pathways in T cell co‐stimulation and autoimmune disease are incompletely understood. We sought to address this issue by investigation of the genesis of acetylcholine receptor (AChR)‐induced antibody‐mediated experimental autoimmune myasthenia gravis (EAMG) in CD28‐ and CD40L‐deficient mice (CD28^{− / −} , CD40L^{− / −} ). Compared to wild‐type mice, the CD28^{− / −} mice became less susceptible, and CD40L^{− / −} mice were completely resistant to EAMG induction. Analysis of T helper functions, reflected by cytokine responses, revealed a switch to a Th1 profile in CD28^{− / −} mice. Consistently, levels of serum AChR‐specific antibodies of the IgG1 isotype were decreased in CD28^{− / −} mice. In the CD40L^{− / −} mice, both Th1 and Th2 cytokine responses were diminished, and T cell‐dependent AChR‐reactive B cell responses were more severely impaired than in the CD28^{− / −} mice. Thus, CD28 and CD40L are differentially required for induction of EAMG.