The thymus is colonized by circulating progenitor cells that differentiate into mature T cells under the influence of the thymic microenvironment. We report here the cloning and function of the avian thymocyte Ag ChT1, a member of the Ig superfamily with one V-like and one C2-like domain. ChT1-positive embryonic bone marrow cells coexpressing c-kit give rise to mature T cells upon intrathymic cell transfer. ChT1-specific Ab inhibits T cell differentiation in embryonic thymic organ cultures and in thymocyte precursor cocultures on stromal cells. Thus, we provide clear evidence that ChT1 is a novel Ag on early T cell progenitors that plays an important role in the early stages of T cell development.