The retinoblastoma gene, RB1, is one of the most frequently mutated genes in human cancer. Rb heterozygous mice develop pituitary tumors with 100% incidence, and the E2F transcription factors are required for this. To assess whether deregulated E2F activity is sufficient to induce pituitary tumors, we generated transgenic mice expressing an inducible E2F3 protein in the intermediate lobe of the pituitary gland. We found that short-term deregulation of E2F activity, similar to the earliest stages of Rb loss, is able to induce abnormal proliferation of otherwise quiescent melanotrophs. However, while long-term exposure to deregulated E2F activity results in hyperplasia of the intermediate lobe, it did not lead to tumor formation. In fact, melanotrophs become insensitive to sustained E2F stimulation and enter an irreversible senescence-like state. Thus, although deregulated E2F activity results in hyperproliferation, it is not sufficient to mimic loss of Rb, sustain proliferation of melanotrophs, and ultimately induce pituitary tumors. Similarly, we found that primary cells in tissue culture become insensitive to sustained E2F3 activation and undergo premature senescence in a pRB-, p16Ink4a-, and p19Arf-dependent manner. Thus, we conclude that deregulated E2F activity is not sufficient to fully mimic loss of Rb due to the engagement of a senescence response.