Mutations in rank among the most common molecular aberrations in human cancer. However, oncogenic consequences of mutation in human cells remain poorly defined due to lack of forward genetic models. Here, CRISPR/Cas9-mediated knockout (KO) in primary human gastric organoids induced morphologic dysplasia, tumorigenicity, and mucinous differentiation. Genetic WNT/β-catenin activation rescued mucinous differentiation, but not hyperproliferation, suggesting alternative pathways of KO-mediated transformation. mutation induced transcriptional regulatory modules characteristic of microsatellite instability and Epstein-Barr virus-associated subtype human gastric cancer, including -associated mitotic genes and survivin. Convergently, high-throughput compound screening indicated selective vulnerability of -deficient organoids to inhibition of BIRC5/survivin, functionally implicating this pathway as an essential mediator of KO-dependent early-stage gastric tumorigenesis. Overall, we define distinct pathways downstream of oncogenic mutation, with nonessential WNT-inhibited mucinous differentiation in parallel with essential transcriptional -stimulated proliferation, illustrating the general utility of organoid-based forward genetic cancer analysis in human cells. SIGNIFICANCE: We establish the first human forward genetic modeling of a commonly mutated tumor suppressor gene, . Our study integrates diverse modalities including CRISPR/Cas9 genome editing, organoid culture, systems biology, and small-molecule screening to derive novel insights into early transformation mechanisms of -deficient gastric cancers...