T-box (Tbx) proteins are dynamically expressed throughout embryogenesis and appear to have significant roles in a number of developmental processes. The T-box family is characterized by a highly conserved DNA binding domain. Over twenty mammalian T-box proteins have been identified, many of which have the demonstrated ability to regulate transcription. Both activators and repressors of gene transcription have been described within this family, but how their functions are performed is largely unknown;Tbx2 is a powerful transcriptional repressor with functional roles identified in cardiogenesis and limb development. However the functional domain(s) of Tbx2 are poorly characterized. Tbx2 contains a putative repression domain carboxy-terminal to the T-box that is conserved between Tbx3 and ET. Although sufficient for Tbx2 induced repression, the carboxy-terminal domain did not appear to be necessary for that repression. A second repression domain was identified at the amino-terminus of Tbx2. In addition, a small activation domain was identified within the amino-terminal portion of the T-box. Characterization of the Tbx2 protein, therefore, identified and delineated multiple functional domains;T-box proteins can bind the same consensus site (TCACAC) and individual functions are believed to depend on specific protein-protein interactions. Surprisingly, few Tbx-protein interactions have been identified. Seven proteins that interacted with Tbx2 were identified from a yeast 2-hybrid screen of an embryonic day 9.5 whole mouse library including: co-activator of activating protein and estrogen receptors (Caper-alpha), ubiquitin specific processing protease 1 (Usp1), heterogeneous nuclear ribonucleoprotein H1, ribosomal protein L5, nascent polypeptide-associated complex alpha, proteasomal subunit (macropain) ATPase3, and E3 ubiquitin ligase HECT domain containing 1. Tbx2 also interacted with Usp1 and Caper-alpha in a mammalian system, as demonstrated by co-immunoprecipitation analysis. Furthermore, areas of co-expression were identified in vivo where Tbx2 could potentially interact with both Usp1 and Caper-alpha;To better understand the roles of Tbx2 in embryogenesis, the functions of Tbx2 needed to be clarified. Repression by Tbx2 has been demonstrated several times in the literature, but a mechanism for how Tbx2 repressed transcription was unknown. The characterization of Tbx2 and identification of Tbx2-protein interactions will help to further our understanding of Tbx2 functions in embryonic development.