Abstract Tumor suppressors control critical steps of cellular metabolism and its inactivation induces cancer specific-metabolic features including aerobic glycolysis. Glycolysis contributes to energy generation and supplying precursors of biomass, e.g., nucleotide, amino sugar, amino acid and lipids. These macromolecules were required for proliferation and cancer survival. Although aerobic glycolysis is now widely accepted as a metabolic feature of cancer, its causal relationship with cancer progression is still unclear. In common type cancers, inactivation of RB tumor suppressor is prevalently observed during tumor progression. Previous studies demonstrated RB inactivation induces inflammation, angiogenesis, drug resistance, etc., as well as cell cycle progression. We currently focus on the impact of RB deficiency on the central carbon metabolism, e.g., glycolysis, TCA cycle and glutamine metabolism. To address this point, we established a mouse model in which RB depletion in p53-null soft tissue sarcoma cells induced undifferentiated phenotype associated with “glycolytic to glutaminolytic” switch (metabolic rewiring) most probably due to down-regulation of Pgam2 activity. Up-regulation of Pgam2 in these cells antagonized the metabolic rewiring, and suppressed spherogenic and tumorigenic activities of RB deficient cells, however did not affect cell proliferation in 2D culture conditions. Moreover, Pgam2 depletion suppressed RB-dependent myogenic differentiation. These findings suggest that RB controls tumorigenicity and differentiation by influencing central carbon metabolism via Pgam2. Citation Format: Susumu Kohno, Nobuyuki Okahashi, Shunsuke Kitajima, Sawako Suzuki, Tomoaki Tanaka, Fumio Matsuda, Hiroshi Shimizu, Chiaki Takahashi. Metabolic rewiring in Rb deficient cells during cancer progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 40.