Molecular diversity via alternative splicing is important for cellular function and development. SR proteins are strong candidate regulators of alternative splicing because they can modulate splice site selection. However, endogenous substrates for SR proteins are largely unknown, and their roles as splicing regulators in vertebrate development are unclear. Here we report that Cre-mediated conditional deletion of the prototypical SR protein SC35 in the thymus causes a defect in T cell maturation. Deletion of SC35 alters alternative splicing of CD45, a receptor tyrosine phosphatase known to be regulated by differential splicing during thymocyte development and activation. This study establishes a model to address the function of SR proteins in physiological settings and reveals a critical role of SC35 in a T cell-specific regulated splicing pathway.