The transmembrane protein tyrosine phosphatase CD45 is differentially required for the development and function of B, T, and NK cells, with mice partially deficient for CD45 having a significant inhibition of T cell, but not NK or B cell, development. CD45-mediated signaling has also been implicated in the development of intrathymic, but not extrathymic, intestinal intraepithelial T lymphocytes (iIELs) in the CD45ex6-/- mouse. As NK1.1+ CD3+ (NK-T) cells can also develop through extrathymic pathways, we have investigated the role of CD45 in NK-T cell development. In mice with a complete absence of CD45 expression (CD45ex9-/-) the NK-T cell population was maintained in the iIEL compartment, but not in the spleen. Functionally, CD45-deficient NK-T cells were unable to secrete IL-4 in response to TCR-mediated signals, a phenotype similar to that of CD45-deficient iIELs, in which in vitro cytokine production was dramatically reduced. Using the CD45ex9-/- mouse strain, we have also demonstrated that only one distinct population of NK-T cells (CD8+) appears to develop normally in the absence of CD45. Interestingly, although an increase in cytotoxic NK cells is seen in the absence of CD45, these NK calls are functionally unable to secrete IFN-γ. In the absence of CD45, a significant population of extrathymically derived CD8αα+ iIELs is also maintained. These results demonstrate that in contrast to conventional T cells, CD45 is not required during the development of CD8+ NK-T cells, NK cells, or CD8αα+ iIELs, but is essential for TCR-mediated function and cytokine production.