Classic T cell subsets are defined by a small set of cell surface markers, while single-cell RNA sequencing (scRNA-seq) clusters cells using genome-wide gene expression profiles. The relationship between scRNA-seq clustered populations (scCPops) and cell surface marker–defined classic T cell subsets remains unclear. In this article, we integrated six bead-enriched T cell subsets with 62,235 single-cell transcriptomes from human PBMCs and clustered them into nine scCPops. Bead-enriched CD4+/CD45RA+/CD25− naive T and CD8+/CD45RA+ naive T cells were mainly clustered into their scCPop counterparts, while cells from the other T cell subsets were assigned to multiple scCPops, including mucosal-associated invariant T cells and NKT cells. The multiple T cell subsets forming one scCPop exhibit similar expression patterns, but not vice versa, indicating scCPop is a more homogeneous cell population with similar cell states. Interestingly, we discovered and named IFN signaling–associated gene (ISAG) high T (ISAG hi T) cells, a T cell subpopulation that highly expressed ISAGs. We further enriched ISAG hi T cells from human PBMCs by FACS of BST2 for scRNA-seq analyses. The ISAG hi T cell cluster disappeared on t-distributed stochastic neighbor embedding plot after removing ISAGs, whereas the ISAG hi T cell cluster showed up by analysis of ISAGs alone, indicating ISAGs are the major contributor of the ISAG hi T cell cluster. BST2+ and BST2− T cells showing different efficiencies of T cell activation indicate that a high level of ISAGs may contribute to quick immune responses.