Harsh environments in poorly perfused tumor regions may select for traits driving cancer aggressiveness. Here, we investigated whether tumor acidosis interacts with driver mutations to exacerbate cancer hallmarks. We adapted mouse organoids from normal pancreatic duct (mN10) and early pancreatic cancer (mP4, KRAS-G12D mutation, ± p53 knockout) from extracellular pH 7.4 to 6.7, representing acidic niches. Viability was increased by acid adaptation, a pattern most apparent in wild-type (WT) p53 organoids, and exacerbated upon return to pH 7.4. This led to increased survival of acid-adapted organoids treated with gemcitabine and/or erlotinib, and, in WT p53 organoids, acid-induced attenuation of drug effects. New genetic variants became dominant during adaptation, yet they were unlikely to be its main drivers. Transcriptional changes induced by acid and drug adaptation differed overall, but acid adaptation increased the expression of gemcitabine resistance genes. Thus, adaptation to acidosis increases cancer cell viability after chemotherapy.