The major aim of this project is to elucidate the mechanisms controlling cell fate decisions in developing T cells. Precursor T cells undergo a testing process in the thymus to ensure that those cells expressing useless or self-reactive T cell antigen receptors (TCR) do not mature (positive and negative selection). These selection processes require TCR engagement of self MHC antigens, but different aspects of these interactions determine whether the cells will live or die. TCR signals promote maturation and also the development of lineages. Precursor thymocytes first must specify the alpha-beta (ab) or gamma-delta (gd) T cell fate. Those that develop in the ab pathway must then choose whether to become CD4 helper or CD8 cytotoxic T cells. Our goal is to understand how TCR signals, acting in concert with other developmental cues, are linked to the process of lineage commitment. In our previous work, we found that the involvement of the CD4 and CD8 coreceptors in TCR recognition of MHC imposes a bias on the CD4 vs. CD8 lineage decision. This most likely occurs through differential recruitment of the tyrosine kinase, LCK, to the coreceptors during thymic selection. We have investigated the role of proximal TCR signaling as well as pathways downstream of the TCR, in efforts to determine how MHC recognition is linked to lineage commitment. We find that deficiencies of ZAP-70 and Tec family tyrosine kinases favor a CD8 over a CD4 fate, lending support to the hypothesis that quantitative differences in TCR signaling can instruct cell fate decisions in developing thymocytes. Similarly, we find that quantitative differences in TCR signaling influence the gd versus ab lineage decision. Increased TCR signaling induced by the over-expression of zeta or by deficiencies of CD5 favors gd at the expense of ab T cell development. Even over-expression of the pre-T alpha (pTa) chain (a component of the pre-TCR) favors a gd over an ab lineage choice, reinforcing the notion that high TCR signals promote the gd lineage decision. Signals through the highly conserved transmembrane receptor, Notch, also influence the gd/ab and CD4/CD8 cell fate decisions in developing T cells. We find that a constitutively active form of Notch can override the bias normally imposed by specific TCR signals, suggesting that these two signaling systems may act in concert to specify cell fate. In other systems, Notch activity can be regulated by ligand binding or by association with other proteins like Numb and Fringe that inhibit responses to Notch ligands. In this regard, we observe that an isoform of Numb is differentially regulated in CD4 and CD8 lineage thymocytes. Of significance, this isoform of Numb has the potential to link the Notch and TCR signaling pathway since it contains a number of SH3 binding domains. In further efforts to understand how Notch signaling is regulated in the thymus, we have studied the Presenilin proteins (PS1/2) that are involved in generating the active form of Notch. We find that dominant negative forms of PS1/2 block early T cell development in the thymus while seemingly promoting the ectopic development of B cells. Thus, Notch appears to regulate lineage decisions at several stages of development including T/B lineage determination.