Most glycolytic enzymes are transcriptionally controlled by hypoxia-inducible factor-1α (HIF-1α) and/or MYC, however, phosphoglycerate mutases (PGAMs) are exceptional. Retinoblastoma tumor suppressor 1 (RB1) loss converts poorly spherogenic Trp53-null leiomyosarcoma cells to highly spherogenic. We determined a gene expression signature of RB1 loss-of-function in this setting and identified PGAM2 as a positive transcriptional target of RB1. Later, we found that RB1 positively controls PGAM1 as well in different tissues. RB1 deficiency induced a metabolic shift in the glycolytic pathway in a manner compatible with PGAM downregulation. Many of the metabolic features induced by RB1 loss were antagonized by PGAM overexpression. Furthermore, differentiation deficiency following RB1 loss was rescued by PGAM overexpression or pyruvate supplementation to varied degrees. These findings suggest that the RB1-PGAM1/2 axis at least partially controls RB1-dependent differentiation.