TheRB1tumor suppressor is recurrently mutated in a variety of cancers including retinoblastomas, small cell lung cancers, triple-negative breast cancers, prostate cancers, and osteosarcomas. Finding new synthetic lethal (SL) interactions withRB1could lead to new approaches to treating cancers with inactivatedRB1. We identified 95 SL partners ofRB1based on aDrosophilascreen for genetic modifiers of the eye phenotype caused by defects in theRB1ortholog,Rbf1. We validated 38 mammalian orthologs ofRbf1modifiers as RB1 SL partners in human cancer cell lines with defectiveRB1alleles. We further show that for many of theRB1SL genes validated in human cancer cell lines, low activity of the SL gene in human tumors, when concurrent with low levels ofRB1was associated with improved patient survival. We investigated higher order combinatorial gene interactions by creating a novelDrosophilacancer model with co-occurringRbf1,PtenandRasmutations, and found that targeting RB1 SL genes in this background suppressed the dramatic tumor growth and rescued fly survival whilst having minimal effects on wild-type cells. Finally, we found that drugs targeting the identified RB1 interacting genes/pathways, such as UNC3230, PYR-41, TAK-243, isoginkgetin, madrasin, and celastrol also elicit SL in human cancer cell lines. In summary, we identified several high confidence, evolutionarily conserved, novel targets forRB1-deficient cells that may be further adapted for the treatment of human cancer.