The accurate distribution of genetic information (chromosomes) during cell division is essential for the growth and proliferation of all living organisms. Errors in chromosome segregation in humans have been linked to cancer progression, infertility and developmental diseases. In my PhD I study how chromosome segregation is regulated in the genetically amenable budding yeast Saccharomyces cerevisiae. Since the mechanisms of chromosome segregation are highly conserved amongst eukaryotes, studies in yeast will provide a fundamental understanding of this process. Sgo1 is the budding yeast member of a highly conserved family of shugoshin proteins, which play a key role in chromosome segregation. My work characterizes a previously unidentified role of Sgo1 in inhibiting separase; an enzyme that triggers chromosome segregation by cleaving the cohesin protein complex that holds replicated chromosomes together. I demonstrate that this novel function of Sgo1 requires a specific form of Protein Phosphatase 2A (PP2ACdc55), an enzyme that itself is highly conserved amongst eukaryotes. I propose that PP2ACdc55 is a separase inhibitor that is employed by Sgo1 when sister chromatids are not under tension. Finally, I go on to initiate preliminary studies into the mechanism whereby PP2ACdc55 inhibits separase. In sum, this study uncovers an additional layer of separase regulation mediated by Sgo1 and PP2ACdc55 and therefore makes a significant contribution to our understanding of the all-or-nothing nature of chromosome segregation.