The cohesin complex comprises four core subunits: RAD21, SMC3, SMC1A, and STAG2. Cohesin is required for many biological processes, including sister chromatid cohesion, DNA damage response, and ribosomal biogenesis. Somatic mutations in cohesin subunits are found in a wide range of human cancers, including acute myeloid leukemia (AML) (12%), Ewing sarcoma (15%) and glioblastoma (19%). Currently, there is no targeted therapeutic strategy for cancer patients with cohesin mutations. Synthetic lethality has emerged as a promising approach for targeted therapy. To identify synthetic lethal drug activity with cohesin mutations, we generated three isogenic cohesin-deficient MCF10A cell lines (RAD21+/-, SMC3+/-, and STAG2-/-) using CRISPR/Cas9. Cohesin-deficient MCF10A cell lines exhibit alterations in nucleolar morphology and increased γH2AX and p53 (except in STAG2-/-). RNA-sequencing analysis reveals downregulation of genes involved in regulation of RNA synthesis in these cell lines. Furthermore, ribosomal stress mediated by Actinomycin D led to severe nucleolar fragmentation in cohesin-deficient MCF10A, but not MCF10A parental, suggesting that cohesin mutation might confer vulnerability to perturbation of ribosome biogenesis. Using cohesin-deficient MCF10A cell lines, we performed a high-throughput drug screen against 3,009 compounds of FDA-approved drugs, kinase, and epigenetic inhibitors. Several classes of inhibitors targeting mTOR signaling, Wnt signaling and chromatin modification were found to selectively inhibit the growth of cohesin-deficient MCF10A cells by 30% or more. In summary, we have identified potential synthetic lethal compounds that may provide the basis for development of targeted therapies of cancers with cohesin deficiency or mutation.