Nucleophosmin (NPM or B23) plays key roles in ribosome biogenesis, centrosome duplication, and maintenance of genomic integrity. Mutations affecting the carboxylterminal domain of NPM occur in a significant percentage of adult patients with acute myeloid leukemia (AML), and these alterations create an additional nuclear export signal that relocalizes much of the protein from its normal nucleolar stores to the cytoplasm. When induced by oncogenic stress, the Arf tumor suppressor protein accumulates within the nucleolus, where it is physically associated with, and stabilized by, NPM. Ectopic overexpression of an NPM cytoplasmic mutant (NPMc) relocalized p19Arf and the endogenous NPM protein to the cytoplasm. NPMc-dependent export of p19Arf from the nucleus inhibited its functional interaction with the p53 negative regulator, Mdm2, and blunted Arf-induced activation of the p53 transcriptional program. Cytoplasmic NPM relocalization also attenuated Arf-induced sumoylation of Mdm2 and NPM and prevented wild type NPM from inhibiting p19Arf protein turnover. However, despite the ability of NPMc to interfere with these p53-dependent and independent activities of Arf, NPMc exhibited anti-proliferative activity in Arf-null NIH-3T3 cells. Overexpression of wild type NPM, but not NPMc, overcame premature senescence of Atm-null cells, a phenotype that can be rescued by inactivation of Arf or p53. Therefore, perturbation of Arf function appears to be insufficient to explain the oncogenic effects of the NPMc mutation. We favor the idea that NPMc also contributes to AML by dominantly perturbing other functions of the wild type NPM protein.