The recognition by the T cell receptor (TCR) of self-peptides presented by the major histocompatibility complex (MHC) on antigen-presenting cells, such as dendritic cells and thymic epithelial cells, controls T cell fate in the thymus, with weak TCR signals inducing survival (positive selection) and stronger signals inducing death (negative selection). In vitro studies indicate that peptide ligands that induce positive selection stimulate a low, but sustained, pattern of TCR signaling; however, the temporal pattern of TCR signaling in MHC class I-restricted thymocytes (thymocytes that are presented with peptides by MHC class I) in the thymus, under conditions that support positive selection, is unknown. We addressed this question by examining intracellular Ca2+ dynamics and migratory changes in thymocytes undergoing positive and negative selection in thymic slices. Brief, serial signaling events that were separated by migratory periods and low cytosolic Ca2+ concentrations correlated with the positive selection of MHC class I-restricted thymocytes, whereas sustained Ca2+ signaling and the arrest of thymocytes were associated with negative selection. Low-avidity peptides and the presentation of peptides by cortical thymic epithelial cells, rather than dendritic cells, failed to induce strong migratory arrest of thymocytes, which led to transient TCR signaling. Thus, we provide a comparison of positive and negative selection signals in situ and suggest that the absence of strong stop signals distinguishes between positive and negative selection.