(A) Immunoblot for Tdh, H3K4me3, H3K27me3, and pan-methyl-lysine in mESCs grown under 0.1X Thr, immediately after depletion of Tdh with 2 different shRNAs (shTdh), compared to a control shRNA targeting luciferase (shLuc). (B) Micrographs of alkaline-phosphatase-staining in mESCs after shTdh or shLuc, seeded at clonal density without feeder MEFs and after 48h of culture in 0.1X, 0.3X, and 1X Thr concentrations. (C) Steady-state fraction of intracellular metabolites derived from [U-13C]Thr in mESCs after 24h, as measured by SRM analysis, after 48h dox-induction of shTdh or shLuc (n=3). (D) SRM analysis of metabolite abundances in mESCs after 48h dox-induction of shTdh or shLuc (n=3). All error bars represent the s.e.m. from three independent measurements.