Memory and effector CD8 T cells differentiate from a single progenitor cell through differentiation, and clonal expansion with asymmetric division. Despite numerous mouse studies exploring the characteristics and determinants of the diverse T cell fates, comparable knowledge in humans is limited. A novel in vitro single cell colony expansion protocol for generation of human CD8+ T cell clones specific for Cytomegalovirus was established with three donors using cognate peptide, IL-2/IL-15 and autologous B-LCLs for antigen presentation. Single TSCM progenitor cells had superior colony forming capacity over TCM and TEM, with clonality confirmed by TCR sequencing. Phenotypic and functional characterisation of the single TSCM-derived progeny revealed differentiated memory and effector subsets (TCM, TEM, TEFF) and also TSCM, with the latter subset featuring greater multipotency and multiple cytokine production. Single cell transcriptomic analysis of the varied progeny cells (n=509) revealed strong “progenitor-progeny” effects, reflecting heritable genetic traits specific to subjects and progenitors rather than progeny phenotypes. Nevertheless, the progeny formed distinct transcriptomic clusters with genes such as Sell, Tef1, and Il7r expressed only in the TSCM-derived progeny (n=13 clones; n= 231 cells). Further analysis with the transcriptomic dataset partitioned by: subject, phenotype of the progenitor, and by individual clone, revealed a unique molecular signature and enriched gene sets shared by TSCM-derived TSCM-progeny cells across subjects and clones. The TSCM signature included both previously reported and novel genes and signalling pathways. These data will inform studies seeking to induce TSCM cells by immunisation or utilise them in immunotherapy.