AbstractAnticancer drugs are at the frontline of cancer therapy. However, innate resistance to these drugs occurs in one‐third to one‐half of patients, exposing them to the side effects of these drugs with no meaningful benefit. To identify the genes and pathways that confer resistance to such therapies, we performed a genome‐wide screen in haploid human embryonic stem cells (hESCs). These cells possess the advantage of having only one copy of each gene, harbour a normal karyotype, and lack any underlying point mutations. We initially show a close correlation between the potency of anticancer drugs in cancer cell lines to those in hESCs. We then exposed a genome‐wide loss‐of‐function library of mutations in all protein‐coding genes to 10 selected anticancer drugs, which represent five different mechanisms of drug therapies. The genetic screening enabled us to identify genes and pathways which can confer resistance to these drugs, demonstrating several common pathways. We validated a few of the resistance‐conferring genes, demonstrating a significant shift in the effective drug concentrations to indicate a drug‐specific effect to these genes. Strikingly, the p53 signalling pathway seems to induce resistance to a large array of anticancer drugs. The data shows dramatic effects of loss of p53 on resistance to many but not all drugs, calling for clinical evaluation of mutations in this gene prior to anticancer therapy.