The analysis of synthetic genetic interaction networks can reveal how biological systems achieve a high level of complexity with a limited repertoire of components. Studies in yeast and bacteria have taken advantage of collections of deletion strains to construct matrices of quantitative interaction profiles and infer gene function. Yet comparable approaches in higher organisms have been difficult to implement in a robust manner. Here we report a method to identify genetic interactions in tissue culture cells through RNAi. By performing more than 70,000 pairwise perturbations of signaling factors, we identified >600 interactions affecting different quantitative phenotypes of Drosophila melanogaster cells. Computational analysis of this interaction matrix allowed us to reconstruct signaling pathways and identify a conserved regulator of Ras-MAPK signaling. Large-scale genetic interaction mapping by RNAi is a versatile, scalable approach for revealing gene function and the connectivity of cellular networks.