The cell wall of Mycobacterium tuberculosis is composed of diverse glycolipids which potentially interact with the human immune system. To overcome difficulties in obtaining pure compounds from bacterial extracts, we recently synthesized three forms of mycobacterial diacyltrehalose (DAT) that differ in their fatty acid composition, DAT1, DAT2, and DAT3. To study the potential recognition of DATs by human T cells, we treated the lipid-binding antigen presenting molecule CD1b with synthetic DATs and looked for T cells that bound the complex. DAT1- and DAT2-treated CD1b tetramers were recognized by T cells, but DAT3-treated CD1b tetramers were not. A T cell line derived using CD1b-DAT2 tetramers showed that there is no cross-reactivity between DATs in an IFN-γ release assay, suggesting that the chemical structure of the fatty acid at the 3-position determines recognition by T cells. In contrast with the lack of recognition of DAT3 by human T cells, DAT3, but not DAT1 or DAT2, activates Mincle. Thus, we show that the mycobacterial lipid DAT can be both an antigen for T cells and an agonist for the innate Mincle receptor, and that small chemical differences determine recognition by different parts of the immune system.