During T cell development, the T cell receptors found on the surface of immature thymocytes (T cell precursors) are screened for their ability to recognize peptide-bound major histocompatibility complexes, a process known as positive selection. Additionally, autoreactive thymocytes are eliminated by negative selection to produce a protective, yet self-tolerant, repertoire of T cells. One widely held model of thymocyte selection posits that weak T cell receptor signals promote thymocyte survival and differentiation, whereas stronger signals lead to deletion of the cells by negative selection. Here, we used two-photon microscopy to define T cell receptor signaling during thymocyte selection within intact thymic tissue under conditions that preserved the dynamic migration of thymocytes and their interaction with thymic cells. We found that negative selection was associated with prolonged T cell receptor signaling and stable cellular interactions, whereas positive selection involved surprisingly brief and infrequent T cell receptor signals. We also investigated the contributions of the type of peptide-presenting cells, costimulatory signals, and ligand potency to the pattern of T cell receptor signaling. Our data shed light on how different components of the thymic micro-environment contribute to temporal TCR signaling patterns during positive and negative selection.