Diffuse gastric cancer (DGC) is an aggressive and frequently lethal subtype of gastric cancer (GC). Because DGC often lacks genomic aberrations that indicate clear candidate therapeutic targets, it has been challenging to develop targeted therapies for this gastric cancer subtype. Our previous study highlighted the contribution of focal adhesion kinase (FAK) in the tumorigenesis of DGC and the potential efficacy of small-molecule FAK inhibitors. However, drug resistance to monotherapy often hinders the efficacy of treatment.We generated a genome-scale library of open reading frames (ORF) in the DGC model of Cdh1-/-RHOAY42C/+ organoids to identify candidate mechanisms of resistance to FAK inhibition. Compensatory activated pathways were also detected following treatment with FAK inhibitors. Candidates were investigated by co-targeting in vitro and in vivo experiments using DGC.We found that cyclin-dependent kinase 6 (CDK6) promoted FAK inhibitor resistance in ORF screen. In addition, FAK inhibitor treatment in DGC models led to compensatory MAPK pathway activation. Small molecule CDK4/6 inhibitors or MAPK inhibitors effectively enhanced FAK inhibitor efficacy in vitro and in vivo.Our data suggest that FAK inhibitors combined with MAPK inhibitors or CDK4/6 inhibitors warrant further testing in clinical trials for DGC.