The gut microbiome shapes the host metabolome, both locally in the gastrointestinal tract, and peripherally across systemic tissues. Our labs have previously shown that secondary bile acids (BAs), which are bacterial metabolites of host-derived primary BAs, can modulate host intestinal immunity by altering T cell differentiation in the ileum and colon. We now report that bacterial BA metabolism can influence extraintestinal immune tissue function, focusing on thymic T cell development. In mice, we observe that secondary BAs make up a larger proportion of the thymic bile acid pool compared to other tissues, and reach concentrations that can activate the BA receptor TGR5, which we also find expressed in the thymus. In both loss-of-function and gain-offunction experiments, we show that secondary BAs can regulate thymic expression of the chemokine Ccl21, likely in thymic medullary epithelial cells. As Ccl21 controls progenitor recruitment, thymocyte migration to the medulla for negative selection, and egress of mature T cells, this signaling pathway may have important implications for T cell selection and adaptive immunity more broadly. Together, these data provide evidence of a novel gut-thymus axis mediated by secondary BAs, through which gut bacteria can influence T cell development from its earliest stages.