Nutrient stress in the tumor microenvironment (TME) requires cancer cells to adopt adaptive metabolic programs for survival and proliferation. Therefore, knowledge of microenvironmental nutrient levels and how cancer cells cope with such nutrition is critical to understand the metabolism underpinning cancer cell biology. Characterizing the metabolic adaptations of cancer cells under the nutrient constraints of the TME can lead to the discovery of novel targetable tumor liabilities. However, our ability to study such adaptations has been precluded by our limited understanding of the metabolic make-up of the TME.Tumor interstitial fluid (TIF) is the local perfusate of the TME that carries metabolites, electrolytes, and soluble macromolecules to tumor resident cells. Previously, we performed quantitative metabolomics of the TIF of murine pancreatic ductal adenocarcinoma (PDAC) tumors to comprehensively characterize nutrient availability in the PDAC TME. In this dissertation, we develop Tumor Interstitial Fluid Medium (TIFM), a cell culture medium that contains nutrient levels representative of the PDAC microenvironment, enabling us to study PDAC metabolism ex vivo under physiological nutrient conditions. We show that PDAC cells cultured in TIFM adopt a cellular state closer to that of PDAC cells present in tumors compared to standard culture models. Further, using the TIFM model, we found arginine biosynthesis is active in PDAC and allows PDAC cells to maintain levels of this amino acid despite microenvironmental arginine depletion. We also show that myeloid-derived arginase activity is largely responsible for the low levels of arginine in PDAC tumors. Altogether, these data indicate that nutrient availability in tumors is an important determinant of cancer cell metabolism and behavior, and cell culture models that incorporate physiological nutrient availability have improved fidelity to in vivo systems and enable the discovery of novel cancer metabolic phenotypes.