Centromere protein F (CENP-F) (or mitosin) accumulates to become an abundant nuclear protein in G2, assembles at kinetochores in late G2, remains kinetochore-bound until anaphase, and is degraded at the end of mitosis. Here we show that the absence of nuclear CENP-F does not affect cell cycle progression in S and G2. In a subset of CENP-F depleted cells, kinetochore assembly fails completely, thereby provoking massive chromosome mis-segregation. In contrast, the majority of CENP-F depleted cells exhibit a strong mitotic delay with reduced tension between kinetochores of aligned, bi-oriented sister chromatids and decreased stability of kinetochore microtubules. These latter kinetochores generate mitotic checkpoint signaling when unattached, recruiting maximum levels of Mad2. Use of YFP-marked Mad1 reveals that throughout the mitotic delay some aligned, CENP-F depleted kinetochores continuously recruit Mad1. Others rebind YFP-Mad1 intermittently so as to produce 'twinkling', demonstrating cycles of mitotic checkpoint reactivation and silencing and a crucial role for CENP-F in efficient assembly of a stable microtubule-kinetochore interface.