Phthiocerol dimycocerosate (PDIM) is an essential virulence lipid of Mycobacterium tuberculosis. In vitro culturing rapidly selects for spontaneous mutations that cause PDIM loss leading to virulence attenuation and increased cell wall permeability. We discovered that PDIM loss is due to a metabolic deficiency of methylmalonyl-CoA that impedes the growth of PDIM-producing bacilli. This can be remedied by supplementation with odd-chain fatty acids, cholesterol, or vitamin B12. We developed a much-needed facile and scalable routine assay for PDIM production and show that propionate supplementation enhances the growth of PDIM-producing bacilli and selects against PDIM-negative mutants, analogous to in vivo conditions. Our results solve a major issue in tuberculosis research and exemplify how discrepancies between the host and in vitro nutrient environments can attenuate bacterial pathogenicity.