Tumorigenic pituitary tumor transforming gene (PTTG) is a mammalian homolog of Xenopus securin that inhibits chromatid separation, is overexpressed in many human tumor types, and mediates transcriptional activation. Loss of yeast securin Pds1p or Drosophila securin pimples is lethal. Here we show that mice lacking PTTG (PTTG −/−) are, surprisingly, viable and fertile; but they have testicular and splenic hypoplasia, thymic hyperplasia, and thrombocytopenia. PTTG −/− mouse embryo fibroblasts exhibited aberrant cell cycle progression with prolonged G₂-M phase and binucleated and multinucleated nuclei with increased aneuploidy. PTTG −/− mouse embryo fibroblast metaphases contained quadriradial, triradial, and chromosome breaks, as well as premature centromere division. The results show that PTTG functions to maintain chromosome stability, cell cycle progression, and appropriate cell division. Moreover, mammalian sister chromatid separation, an important transition in the cell cycle, is likely regulated by mechanisms in addition to securin.