Synthetic chromosomes have the potential to revolutionize gene therapy, because they can deliver entire genetic programs. However, this strategy can succeed only if the synthetic chromosomes are faithfully transmitted to each daughter cell upon cell division. Eukaryotic cells use the kinetochore, an immensely complex protein machine, to segregate their native chromosome during cell division. We are initiating an effort to engineer a synthetic kinetochore to faithfully segregate synthetic chromosomes. This ambitious goal encompasses three aims. First, we will use bottom-up reverse engineering to yield deep insight into function and regulation of the kinetochore, potentially exposing unexpected gaps in our understanding. Second, we will identify functionally necessary kinetochore components from those that are its ‘evolutionary legacy’. This is necessary to reveal strategies for simplifying the design of the synthetic kinetochore. Third, we will establish the blueprint for designing a synthetic, orthogonal segregation machine that specifically controls the inheritance of synthetic chromosomes.