TNF family members play crucial roles in mammalian B-cell differentiation and function, many of which have not been demonstrated in other species. To investigate the avian CD40/CD40L system, a chicken CD40 cDNA, obtained by expression screening, was used to raise monoclonal antibodies showing that CD40 was expressed on chicken B cells, monocytes and macrophages, like mammalian CD40. CD40 ligand fusion protein supported the proliferation of B cells in culture for up to 3 weeks, during which they differentiated towards a plasma cell phenotype. CD40L-activated B cells from immunised birds secreted antigen-specific IgM and IgG. These results showed important conserved functions of CD40 and its ligand in mammals and birds. CD40L provides a means for maintenance and differentiation of untransformed chicken B cells in culture, for the first time, allowing new approaches to study of post-bursal B cell biology and host-pathogen interactions with B cell tropic viruses.