The Wiskott-Aldrich syndrome protein (WASP) is a key cytoskeletal regulator of hematopoietic cells. Although WASP-knockout (WKO) mice have aberrant B-cell cytoskeletal responses, B-cell development is relatively normal. We hypothesized that N-WASP, a ubiquitously expressed homolog of WASP, may serve some redundant functions with WASP in B cells. In the present study, we generated mice lacking WASP and N-WASP in B cells (conditional double knockout [cDKO] B cells) and show that cDKO mice had decreased numbers of follicular and marginal zone B cells in the spleen. Receptor-induced activation of cDKO B cells led to normal proliferation but a marked reduction of spreading compared with wild-type and WKO B cells. Whereas WKO B cells showed decreased migration in vitro and homing in vivo compared with wild-type cells, cDKO B cells showed an even more pronounced decrease in the migratory response in vivo. After injection of 2,4,6-trinitrophenol (TNP)-Ficoll, cDKO B cells had reduced antigen uptake in the splenic marginal zone. Despite high basal serum IgM, cDKO mice mounted a reduced immune response to the T cell-independent antigen TNP-Ficoll and to the T cell-dependent antigen TNP-keyhole limpet hemocyanin. Our results reveal that the combined activity of WASP and N-WASP is required for peripheral B-cell development and function.