Mutations in Bruton's tyrosine kinase (Btk) result in the B-cell immunodeficiencies X-linked agammaglobulinemia in humans and X-linked immunodeficiency in mice. These diseases are characterized by blocks in B-cell development at multiple stages and impaired function of residual mature B cells. This review focuses on a series of in vivo genetic studies that have begun to define the mechanism by which Btk regulates B-cell development and function. The functional interactions between Btk and other signaling molecules defined by this approach are more complex than initially appreciated from in vitro biochemical and cell culture studies.