At any given moment, 250 million cells are dividing in the human body through an essential process known as mitosis. Inaccuracy of mitosis leads directly to aneuploidy (gain or loss of chromosomes), a hallmark of several cancers and birth defects. Mitotic fidelity is controlled by the spindle assembly checkpoint (SAC), a signaling pathway that delays the progression of mitosis to ensure that all chromosomes are attached to mitotic spindle microtubules (MTs). Central to this activity, the kinetochore (KT), a minute structure on each replicated sister-chromatid, promotes the rapid turnover of MTs to correct potential attachment errors during early mitotic stages. Upon anaphase onset, the KT then switches to bind MTs with higher affinity, so that the energy derived from their depolymerizing plus ends helps driving chromosome motion to the poles. While the molecular basis of the KT-MT interface is only now starting to be elucidated, how the multiple KT activities are regulated throughout mitosis remains unknown. Here we propose to dissect from a molecular perspective how the interaction between spindle MTs and KTs controls chromosome segregation fidelity in space and time. For this purpose we will combine the power of biochemical analysis and genome-wide RNAi screens with the detailed functional investigation of already identified candidate genes using state-of-the-art live cell microscopy and pilot laser microsurgery tools in animal cells. Additionally, we have in place all the necessary conditions to investigate the physiological significance of chromosome segregation errors and evaluate respective outcomes using unique mammalian model systems. With this synergistic approach we aim to unveil the molecular routes of aneuploidygenesis and their implications to human health.