Inactivation of multiple endocrine neoplasia (MEN) type 1 gene (Men1) results in development of multiple endocrine tumors in Men1(+/-) mice and in humans. Intriguingly, loss of the wild-type retinoblastoma 1 (Rb) gene also leads to MEN-like phenotype in Rb(+/-) mice. To evaluate potential genetic interactions between these genes, we prepared and characterized Men1(+/-)Rb(+/-) compound mice in parallel with their parental genotypes. Men1 and Rb did not cooperate in tumor suppression, as demonstrated by comparable survival rates of Rb(+/-) and Men1(+/-)Rb(+/-) mice, absence of tumor growth acceleration and lack of novel neoplasms. Notably, the loss of the remaining copy of the wild-type Men1 and Rb was mutually exclusive in all tumors of Men1(+/-)Rb(+/-) mice, including pituitary anterior lobe and adrenal medulla neoplasms shared by Rb- and Men1-deficient phenotypes. Down-regulation of Men1 targets p18 and p27 and increased presence of phosphorylated-Rb were observed in Men1-deficient pheochromocytomas of Men1(+/-)Rb(+/-) and Men1(+/-) mice. At the same time, the RNA interference (RNAi) knock-down of Men1 mRNA resulted in increased apoptosis of Rb-deficient medullary thyroid carcinoma cells. These results demonstrate that, depending on cell lineage context, combined Men1 and Rb deficiency may be either redundant or detrimental to neoplastic growth. Identification of cell lineage-specific interactions between Men1 and Rb may have important implications for development of rationally designed therapeutic approaches.