Recent in vitro studies have shown molecular and functional disruptions in RB1 gene heterozygous (RB1+/−) fibroblasts and mesenchymal stem cells (MSCs), even without biallelic loss of RB1. However, this was not observed in RB1+/− retinal organoids. To understand these differences, researchers conducted high-throughput RNA sequencing analysis on induced pluripotent stem cells (iPSCs) derived from RB1+/+ and RB1+/− retinoblastoma patients. They subjected these iPSCs to retinal differentiation and evaluated the process using molecular and flow cytometry analysis. RNA sequencing revealed a metabolic shift from glycolysis to oxidative phosphorylation in RB1+/− retinal organoids. Further assays demonstrated increased ATP and pyruvate levels in RB1+/− retinal organoids compared to RB1+/+. These findings suggest dysregulation of energy metabolism and glycolytic pathways as an early event in RB1 mutation, preceding other phenotypic consequences like altered cellular proliferation.