Pancreatic acinar cells acquire in vitro a pancreatic progenitor phenotype associated with activation of p53, growth arrest and senescence. A similar program is also activated in chronic pancreatitis. To assess the mechanisms involved in this process, we cultured pancreatic acinar cells from wild-type, p53(-/-), p16(-/-) and p21(-/-) mice. Cultures from p53(-/-) mice, but not those from p16(-/-) or p21(-/-) mice, display an enhanced proliferation and can be expanded continuously for more than 20 passages. p53(-/-) cells also display features of stemness such as enhanced sphere formation, increased expression of pancreatic multipotent progenitor markers (Ptf1a, Pdx1, Cpa1, c-myc, Sox9 and Hnf1b), and of the stemness regulators Bmi1 and Klf4. Upon subculture, p53(-/-) cells undergo an epithelial-mesenchymal transition (EMT) and express high levels of vimentin and of the transcriptional regulators Snai1, Snai2, Twist, Zeb1 and Zeb2. Genetic lineage tracing unequivocally demonstrates the epithelial origin of the cells with mesenchymal phenotype. These cells express the endodermal markers Hhex, Pdx1, Sox9, Hnf1b, Foxa2, Gata6 and Sox17, and the stem cell markers c-myc, Bmi1 and Klf4. Cultures from p53(+/-) mice display intermediate levels of the transcription factors involved in EMT but do not surpass the growth arrest. Our findings support the notion that p53 controls both growth and epithelial cell differentiation in the pancreas. These observations have important implications regarding the mechanisms through which p53 inactivation in tumors may be associated with aggressive biological behavior.