Oesophageal cancer is the 8th most common cancer worldwide and has the 6th highest mortality rate of all cancers. The 5-year survival rate following oesophageal adenocarcinoma (OAC) diagnosis is dismal at <15%, indicating a dire need for improved therapeutic strategies and early detection. OAC develops stepwise following exposure to chronic gastric reflux: From pre-malignant Barrett’s metaplasia through low- and high-grade dysplasia to invasive cancer. Mutation or loss of common tumour suppressor genes TP53 and SMAD4 act as markers for cancer progression, occurring in high-grade dysplastic tissue and invasive OAC, respectively. Our group have established a novel in vivo tumourigenesis model for progression of Barrett’s metaplasia to OAC, in which SMAD4-deficient Barrett’s metaplasia cells form tumours in immunodeficient mice after a period of latency and in a dose-dependent manner. This delayed tumour growth onset suggests further genomic changes required for oncogenesis. Our finding that increased copy number alterations co-occur with SMAD4 deficiency strengthens this and suggests that SMAD4 may play a role in maintaining genomic stability. This work aimed to identify and validate novel therapeutic targets as well as co-operative drivers of tumourigenesis for SMAD4-deficient oesophageal adenocarcinoma. We have conducted simultaneous genome-wide CRISPR-Cas9 knockout synthetic lethality and tumourigenesis screens in both in vitro and in vivo settings with a background of SMAD4 wildtype and knockout cells. Our in vivo tumourigenesis screen produced tumours 4-fold faster than the previous model and promises to identify cooperative drivers of tumourigenesis in SMAD4-deficient OAC. The synthetic lethality screen has identified numerous potential therapeutic targets, both novel and clinically relevant, for SMAD4-deficient OAC. The identification of genes and pathways that co-operate with SMAD4 deficiency in pre-malignant cells to promote tumourigenesis will allow for improved patient screening and inform future treatment options. Additionally, the identification of genes and pathways that are essential in SMAD4-deficient tumours will unveil new potential treatment targets for SMAD4-deficient cancers.