Alteration of cellular energy metabolism is a principal feature of tumor and stem cells. Here we analyze the metabolic interactions between cancer cells and fibroblasts in a co-culture model and the metabolic heterogeneity of tumors and metabolic changes in mesenchymal stem cells during adipogenic differentiation based on the fluorescence of the metabolic cofactors NADH, NADPH, and FAD. We registered a metabolic switch from oxidative phosphorylation to glycolysis with slight acidification of the cytosol in cancer cells in a co-culture model. In the tumor tissue we detected metabolic heterogeneity with more glycolytic metabolism of cancer cells in the stroma-rich zones. The shift of cellular energy metabolism from glycolysis to oxidative phosphorylation and the activation of lipogenesiswere observed in adipocytes. Data aboutmetabolic alterations in cancer and stemcells are important formonitoring the progression of cancers, the development of anticancer drugs and stemcell therapy.