Cultured cells convert glucose to lactate, and glutamine is the major source of tricarboxylic acid (TCA)-cycle carbon, but whether the same metabolic phenotype is found in tumors is less studied. We infused mice with lung cancers with isotope-labeled glucose or glutamine and compared the fate of these nutrients in tumor and normal tissue. As expected, lung tumors exhibit increased lactate production from glucose. However, glutamine utilization by both lung tumors and normal lung was minimal, with lung tumors showing increased glucose contribution to the TCA cycle relative to normal lung tissue. Deletion of enzymes involved in glucose oxidation demonstrates that glucose carbon contribution to the TCA cycle is required for tumor formation. These data suggest that understanding nutrient utilization by tumors can predict metabolic dependencies of cancers in vivo. Furthermore, these data argue that the in vivo environment is an important determinant of the metabolic phenotype of cancer cells. Display omitted •Metabolic phenotyping of tumors can identify essential metabolic pathways•Kras-driven lung tumors require pyruvate carboxylase and pyruvate dehydrogenase•Kras-driven lung tumors are less dependent on glutaminase than cultured cells•Tissue environment is an important determinant of tumor metabolic phenotypes Davidson et al. find that lung cancer cells in culture use nutrients differently than lung tumors, especially regarding glutamine metabolism. This difference in metabolic phenotype reflects how the tumor environment determines nutrient dependency and highlights the importance of studying cancer metabolism in a physiological context.