It is unknown how the contrasting events of positive and negative selection can lead to the distinct biological outcomes of life or death. An increasing body of evidence suggests that the duration of extracellular signal-regulated kinase (ERK) signaling plays a role in thymocyte selection. However, it remains unclear what the kinetics of ERK activation are during positive selection in vivo. In this study, we examined the magnitude and duration of ERK signaling in intact murine thymic tissues cultured under conditions of negative or positive selection. We found that negative selection induced a rapid and robust ERK activation that is associated with death, whereas positive selection stimulated a lower intensity and brief ERK activation that quickly declined and then gradually increased and was sustained over several days. The expression pattern of Egr-1 (early growth response-1), a downstream ERK effector, correlates with the biphasic kinetics of ERK during positive selection. Id3 (inhibitor of differentiation/DNA binding 3) also exhibits biphasic kinetics but appeared to be independent of ERK signaling. Furthermore, inhibitors of T cell receptor ligation and ERK activation block maturation of CD8 single-positive thymocytes even when added after 24 h. These results demonstrate that the in vivo duration of ERK signaling must be sustained to support positive selection.