Significance Rare human hereditary disorders provide unequivocal evidence of the role of gene mutations in human disease pathogenesis and offer powerful insights into their influence on human disease development. Using a hereditary retinoblastoma (RB) patient–derived induced pluripotent stem cell (iPSC) platform, we elucidate the role of pRB/E2F3a in regulating spliceosomal gene expression. Pharmacological inhibition of the spliceosome in RB1 -mutant cells preferentially increases splicing abnormalities of genes involved in cancer-promoting signaling and impairs cell proliferation and tumorigenesis. Expression of pRB/E2F3a–regulated spliceosomal proteins is negatively associated with pRB expression and correlates with poor clinical outcomes of osteosarcoma (OS) patients. Our findings strongly indicate that the spliceosome is an “Achilles’ heel” of RB1 -mutant OS.