Lnk is an adaptor protein expressed primarily in lymphocytes and hemopoietic precursor cells. Marked expansion of B lineage cells occurs in lnk(-/-) mice, indicating that Lnk regulates B cell production by negatively controlling pro-B cell expansion. In addition, lnk(-/-) hemopoietic precursors have an advantage in repopulating the hemopoietic system of irradiated host animals. In this study, we show that Lnk overexpression results in impaired expansion of lymphoid precursor cells and altered mature B cell subpopulations. The representation of both B lineage and T lineage cells was reduced in transgenic mice overexpressing Lnk under the control of a lymphocyte-specific expression vector. Whereas the overall number of B and T cells was correlated with Lnk protein expression levels, marginal zone B cells in spleen and B1 cells in the peritoneal cavity were relatively resistant to Lnk overexpression. The C-terminal tyrosine residue, conserved among Lnk family adaptor proteins, was dispensable for the negative regulatory roles of Lnk in lymphocyte development. Our results illuminate the novel negative regulatory mechanism mediated by the Lnk adaptor protein in controlling lymphocyte production and function.