Significance Cells generate a vast repertoire of lipid molecules whose functions are poorly understood. To investigate whether lipids can regulate cell fate decisions, we carried out a systematic lipidomic analysis and perturbation of lipid metabolism in cultured human epidermal keratinocytes, determining associations with the onset of differentiation. We identified individual lipid species that induced exit from the epidermal stem cell compartment. Our observations suggest that more research is warranted on the regulation of biological processes via lipid species, moving beyond the more conventional contribution of proteins and nucleic acids.