We show that both genetic inhibition and pharmacologic inhibition of a key enzyme of the mitochondrial arm of the one-carbon metabolic pathway, serine hydroxymethyltransferase 2, lead to substantial inhibition of Ewing sarcoma cell proliferation and colony-forming ability and that this effect is primarily caused by depletion of glycine and one-carbon units required for the synthesis of purine nucleotides. The gene discussed is SHMT2; the disease is Ewing sarcoma.