Diacylated sulfoglycolipids (Ac₂SGL) are a family of cell wall lipids that have only been detected in virulent strains of Mycobacterium tuberculosis. T cells are activated by Ac₂SGL when the lipids are bound to CD1 molecules, but tools to study Ac₂SGL-specific T cell responses in humans are lacking. To study the molecular requirements for T cell receptor (TCR) binding and T cell activation, we generated CD1b tetramers loaded with natural or synthetic Ac₂SGL analogs. Due to the relatively non-polymorphic nature of CD1 among human populations these tetramers can be applied to samples independently of the donor’s genetic background. T cell lines derived using natural Ac₂SGL are activated by synthetic analogs in a manner that is relatively independent of lipid chain length and hydroxylation but sensitive to saturation status. By contrast, two T cell lines derived using a tetramer loaded with unsaturated Ac₂SGL are not activated by the natural antigen. Notably, the TCRs of T cell clones specific for the natural antigen share no similarity to the TCRs of the T cell clones that are only activated by unsaturated Ac₂SGL analogs. As contrasted with other systems in which the naturally occurring lipid variation does not control T cell responses, these data show that each molecular variant within a class of naturally occurring lipids represents a distinct antigen. This principle will guide the emerging use these tetramers in large-scale translational studies investigating the diagnostic potential of SGL-specific T cell responses and SGL-based vaccine strategies. It is imperative to first understand whether synthetic analogs with a precise chemical definition are bioequivalent to the mixture of antigens that comprise natural Ac₂SGL. These data provide a hierarchy of synthetic Ac₂SGL analogs, and analogs with a high bioequivalence to the natural mixture can be used to generate tetramers for such studies.