The adoptive transfer of TCR-transgenic T cells into syngeneic recipients allows characterization of individual T cells during in vivo immune responses. However, the proliferative behavior of individual T cells and its relationship to effector and memory function has been difficult to define. Here, we used a fluorescent dye to dissect and quantify T cell proliferative dynamics in vivo. We find that the average Ag-specific CD4+ T cell that undergoes division in vivo generates> 20 daughter cells. TCR and CD28 signals cooperatively determine the degree of primary clonal expansion by increasing both the proportion of Ag-specific T cells that divide and the number of rounds of division the responding T cells undergo. Nonetheless, despite optimal signaling, up to one-third of Ag-specific cells fail to divide even though they show phenotypic evidence of Ag encounter. Surprisingly, however, transgenic T cells maturing on a RAG-2−/− background exhibit a responder frequency of 95–98% in vivo, suggesting that maximal proliferative potential requires either a naive phenotype or allelic exclusion at the TCRα locus. Finally, studies reveal division cycle-dependent expression of markers of T cell differentiation, such as CD44, CD45RB, and CD62L, and show also that expression of the cytokines IFN-γ and IL-2 depends primarily on cell division rather than on receipt of costimulatory signals. These results provide a quantitative assessment of T cell proliferation in vivo and define the relationship between cell division and other parameters of the immune response including cytokine production, the availability of costimulation, and the capacity for memory.