Faithful transmission of the genome requires that chromosomes are accurately segregated between daughter cells in mitosis. Accurate chromosome segregation relies on proper specification of the site of kinetochore formation on each chromosome, in addition to dynamic regulation of the interactions of the kinetochore with the microtubule-based mitotic spindle. This thesis work focuses on two important contributors to accurate chromosome segregation: 1) centromere protein A (CENP-A), the histone H3 variant which epigenetically specifies the centromere which forms the platform onto which the kinetochore assembles and 2) the chromosomal passenger complex (CPC) which regulates kinetochore-microtubule interactions to ensure accurate genome partitioning between cells. In the first part of this work, we used a combination of in vitro, genomic sequencing, and novel bioinformatic approaches to probe the nature and structure of CENP-A nucleosomes at functional human centromeres. We found that CENP-A exists as part of a stable octameric nucleosome with loose superhelical DNA termini. CENP-A nucleosomes are very highly phased on the α-satellite monomers at normal centromeres and are also strongly positioned at naturally-occurring neocentromeres. In the second part of this work, we used cell biology-based approaches to uncover a novel mechanism to regulate kinetochore-microtubule interactions that was present only in healthy, diploid cells that had been previously overlooked in aneuploid cells. We found that Aurora B, the enzymatic kinase of the CPC, is enriched at the misaligned centromeres of healthy, diploid cells leading to an increased dynamic range of Aurora B substrate phosphorylation at misaligned versus properly aligned kinetochores. These findings suggest that in addition to Aurora B regulating kinetochore-microtubule interactions, the kinetochore also controls Aurora B recruitment to the inner centromere. We showed that this recruitment depends on both the activity of another mitotic kinase, Plk1, in addition to the activity of Aurora B itself. Altogether, this has led us to update the model by which the CPC regulates kinetochore-microtubule interactions on misaligned chromosomes. Taken together, the work presented from both parts of my thesis greatly enhance our understanding of how the kinetochore is specified and regulated to ensure fidelity in genome transmission during cell division.