The mammalian securin, pituitary tumor-transforming gene (Pttg), regulates sister chromatid separation during mitosis. Mice deficient in Pttg expression exhibit organ-specific hypoplasia of the testis, spleen, pituitary, and postmaturity pancreatic beta-cells, pointing to a possible adult stem cell defect. Bone marrow stem cells (BMSCs) contribute to bone, cartilage, and fat tissue repair and regeneration, and multipotent adult progenitor cells (MAPCs) have broader differentiation ability. Bone marrow cells derived under MAPC conditions are involved in a spectrum of tissue repair. We therefore tested whether Pttg deletion affects stem cell proliferation and differentiation. BMSCs were isolated under MAPC conditions, although unlike MAPCs, wild-type (WT) and Pttg(-/-) BMSCs do not express octamer-binding transcription factor 4 and are stem cell antigen-I positive. WT and Pttg(-/-) cells did not differ in their ability to differentiate into adipogenic, osteogenic, or hepatocyte-like cells or in phenotypic markers. Cells underwent >100 population doublings, with no observed transforming events. Pttg-null BMSCs replicated 27% slower than WT BMSCs, and under hypoxic conditions, this difference widened. Although apoptosis was not enhanced in Pttg(-/-) cells, Pttg(-/-) BMSC senescence-associated beta-galactosidase activity was elevated, consistent with enhanced p21 protein levels. Using gene array assays, DNA repair genes were shown to be upregulated in Pttg(-/-) BMSCs, whereas genes involved in cell cycle progression, including cyclin D(1), were decreased. Separase, the protease regulated by Pttg, has been implicated in DNA damage repair and was downregulated in Pttg(-/-) BMSCs. Separase was constitutively phosphorylated in Pttg(-/-) cells, a modification likely serving as a compensatory mechanism for Pttg deletion. The results indicate that Pttg deletion reduces BMSC proliferation, renders cells more sensitive to hypoxia, and enhances senescent features, thus pointing to a role for Pttg in the maintenance and proliferation of BMSCs.