SummaryFactors that govern the complex formation of memory T cells are not completelyunderstood. A better understanding of thedevelopment of memory Tcell hetero-geneity is however required to enhance vaccination and immunotherapy ap-proaches. Here we examined the impact of pathogen- and tissue-specific cueson memory CD8+T cell heterogeneity using high-dimensional single-cell mass cy-tometry and a tailored bioinformatics pipeline. We identified distinct populationsof pathogen-specific CD8+T cells that uniquely connected to a specific pathogenor associated to multiple types of acute and persistent infections. In addition, thetissue environment shaped the memory CD8+T cell heterogeneity, albeit to alesser extent than infection. The programming of memory CD8+T cell differenti-ation during acute infection is eventually superseded by persistent infection.Thus, the plethora of distinct memory CD8+T cell subsets that arise upon infec-tion is dominantly sculpted by the pathogen-specific cues and further shaped by the tissue environment.