The Tec family tyrosine kinases Itk, Tec and Rlk are expressed in T cells. Previous studies have established that these kinases are critical for TCR signaling, leading to the activation of PLCγ1. To further understand the functions of Tec kinases in T cell activation, we took three different approaches. First, we performed a thorough analysis of CD28-mediated signaling events and functional responses with purified naïve T cells from Itk^-/- mice and a highly controlled stimulation system. Data from this set of studies definitively demonstrate that CD28 costimulation functions efficiently in naïve CD4⁺ T cells in the absence of Itk. Second, in order to further study the functions of Tec kinases in vivo, we generated transgenic mouse lines expressing a kinase-dead (KD) mutant of Tec on the Itk^-/-Rlk^-/- background, hoping to study mice that are functionally deficient for all three Tec kinases. The results hint the importance of the Tec kinases in T cell development and/or survival. Finally, in order to identify potential transcriptional targets of Itk, we used microarray technology to compare global gene expression profiles of naïve and stimulated Itk^-/- versus Itk^+/- CD4⁺ T cells. This analysis provided a short list of differentially expressed genes in Itk^-/- versus Itk^+/- CD4 T cells, providing a starting point for further studies of Itk in T cell activation. Collectively, these studies clarified the role of Itk in CD28 signaling, revealed some unexpected aspects of Tec family kinases in T cells, and indicated potential targets of Itk-dependent signaling pathways in T cells.