The kinetochore is structure composed of many proteins that assembles on centromeric DNA to mediate the binding of spindle microtubules to chromosomes and chromosome movement. Budding yeast has been used as a model organism to investgate the mechanisms underlying kinetochore assembly. Although the basic features of chromosome segregation are thought to be common between all eukaryotes, it is difficult to identify components of vertebrate kinetochores by sequence homology with budding yeast kinetochore proteins. Therefore, we must use vertebrate systems to understand the mechanisms of kinetochore assembly and function. Several experimental strategies, including RNA interference (RNAi) in cultured human cells, knockout mice, Drosophila genetics, C. elegans with RNAi, and immunodepletion in Xenopus egg extracts, have been used to examine the mechanisms of kinetochore assembly. Our Lab. is using DT40 cells to identify and characterize kinetochore components. The DT40 system is a powerful and reliable tool for study of kinetochore assembly. Herein, we review recent advances in our understanding of the mechanisms underlying kinetochore assembly in DT40 cells.