The proliferation of T cells is regulated in a development-dependent manner, but it has been unclear whether proliferation is essential for T cell differentiation. The cyclin-dependent kinase inhibitor p27 Kip1 is abundant throughout development in cells of the T cell lineage, with the exception of late stage CD4− CD8− thymocytes and activated mature T cells, both of which show a high rate of proliferation. The role of down-regulation of p27 Kip1 expression in T cell development and function has now been investigated by the generation and characterization of three strains of p27 transgenic mice that express the transgene at various levels specifically in the T cell lineage. The numbers of thymocytes at CD4+ CD8+, CD4+ CD8−, and CD4− CD8+ stages of development as well as those of mature T cells in peripheral lymphoid tissues were reduced in transgenic mice in a manner dependent on the level of p27 Kip1 expression. The development of thymocytes in the transgenic strain in which p27 Kip1 is most abundant (p27-Tg high mice) appeared to be blocked at the CD4− CD8− CD25+ CD44 low stage. Peripheral T cells from p27-Tg high mice exhibited a reduced ability to proliferate in response to mitogenic stimulation compared with wild-type T cells. Moreover, Ag-induced formation of germinal centers and Ig production were defective in p27-Tg high mice. These results suggest that down-regulation of p27 Kip1 expression is required for the development, proliferation, and immunoresponsiveness of T cells.