Maintenance of immunological memory has been proposed to rely on stem-cell-like lymphocytes. However, data supporting this hypothesis are focused on the developmental potential of lymphocyte populations and are thus insufficient to establish the functional hallmarks of stemness. Here, we investigated self-renewal capacity and multipotency of individual memory lymphocytes by in vivo fate mapping of CD8+ T cells and their descendants across three generations of serial single-cell adoptive transfer and infection-driven re-expansion. We found that immune responses derived from single naive T (Tn) cells, single primary, and single secondary central memory T (Tcm) cells reached similar size and phenotypic diversity, were subjected to comparable stochastic variation, and could ultimately reconstitute immunocompetence against an otherwise lethal infection with the bacterial pathogen Listeria monocytogenes. These observations establish that adult tissue stem cells reside within the CD62L+ Tcm cell compartment and highlight the promising therapeutic potential of this immune cell subset. •Individual Tcm cells are multipotent to generate diverse effector and memory subsets•Individual primary Tcm cells can self-renew into multipotent secondary Tcm cells•Responses derived from single Tcm cells show nonheritable stochastic variation•Single secondary Tcm cells can provide full reconstitution of immunocompetence Long-term persistence of adaptive immunity is suggested to rely on stem-cell-based mechanisms. Graef et al. show that upon serial single-cell transfers, propagation of protective T cell memory can be established by individual central memory T cells acting as self-renewing, multipotent tissue stem cells.