Abstract Peripheral CD4+CD8αα+TCRαβ+ intraepithelial lymphocytes (pIELs) are a specialised T cell population that require the presence of intestinal microbes for their development, however the microbes and factors involved are not well characterised. pIELs are closely associated with intestinal epithelial cells (IECs) that line the gastrointestinal tract, but the contribution of IECs in shaping pIEL development is also not well defined. We investigated a number of microbial metabolites for their ability to influence pIEL development, and whether this is mediated by microbe-IEC-pIEL interactions. In this study we utilised flow cytometry to characterise pIEL frequencies in GF mice administered with different microbial metabolites, in SPF mice from different colonies and animal facilities, and in GF mice monocolonised with potential pIEL-inducing commensal species. qPCR of IEC RNA isolated from these mice was performed to identify candidate genes expressed by IECs that may influence pIEL development. We identified a common bacterial metabolite that partially restores pIEL development in GF mice, and a novel pIEL-inducing bacterial species. pIEL frequency of SPF mice varied between different sources, and qPCR identified IECs expressed several T-cell-influencing cytokines that correlated with pIEL frequency in vivo. Our results identify a common microbial metabolite that contributes to pIEL development that may mediate IEC expression of T-cell-influencing cytokines. Our work further elucidates the mechanisms of host tolerance triggered by commensal microbes, which will facilitate the development of better strategies to prevent and treat intestinal infections and inflammatory diseases.