In this study, we use a minimal ex vivo culture system to track CD8 T cell differentiation at the single cell and clonal lineage level during activation. To characterize early heterogeneity in memory and effector differentiation in this system, we subjected cells to temporally-resolved single-cell transcriptomic profiling using sci-fate-seq (Cao et al., Nature Biotech, 202). Overall design: Cells were activated ex vivo and subjected at days 1, 2, and 4 to 4-thiouridine (4sU) pulse-labeling for 2 hrs, enabling detection of newly synthesized mRNA at each time point.