Pituitary tumor transforming gene (Pttg) is induced in pituitary tumors and associated with increased tumor invasiveness. Pttg-null mice do not develop tumors, but exhibit pituitary hypoplasia, whereas mice heterozygous for the retinoblastoma (Rb) deletion develop pituitary tumors with high penetrance. Pttg-null mice were therefore cross-bred with Rb^+/− mice to test the impact of pituitary hypoplasia on tumor development. Before tumor development, Rb^+/−Pttg^−/− mice have smaller pituitary glands with fewer cycling pituitary cells and exhibit induction of pituitary p21 levels. Pttg silencing in vitro with specific short hairpin interfering RNA in AtT20 mouse corticotrophs led to a marked induction of p21 mRNA and protein levels, decreased RB phosphorylation, and subsequent 24% decrease in S-phase cells. Eighty-six percent of Rb^+/−Pttg^+/+ mice develop pituitary adenomas by 13 months, in contrast to 30% of double-crossed Rb^+/−Pttg^−/− animals (P < 0.01). Pituitary hypoplasia, associated with suppressed cell proliferation, prevents the high penetrance of pituitary tumors in Rb^+/− animals, and is therefore a protective determinant for pituitary tumorigenesis.