Haspin phosphorylates histone H3 at threonine-3 (H3T3ph), providing a docking 
site for the Aurora B complex at centromeres. Aurora B functions to correct 
improper kinetochore-microtubule attachments and alert the spindle checkpoint to 
the presence of misaligned chromosomes. We show that Haspin inhibitors decreased 
H3T3ph, resulting in loss of centromeric Aurora B and reduced phosphorylation of 
centromere and kinetochore Aurora B substrates. Consequently, metaphase 
chromosome alignment and spindle checkpoint signaling were compromised. These 
effects were phenocopied by microinjection of anti-H3T3ph antibodies. 
Retargeting Aurora B to centromeres partially restored checkpoint signaling and 
Aurora B-dependent phosphorylation at centromeres and kinetochores, bypassing 
the need for Haspin activity. Haspin inhibitors did not obviously affect 
phosphorylation of histone H3 at serine-10 (H3S10ph) by Aurora B on chromosome 
arms but, in Aurora B reactivation assays, recovery of H3S10ph was delayed. 
Haspin inhibitors did not block Aurora B localization to the spindle midzone in 
anaphase or Aurora B function in cytokinesis. Thus, Haspin inhibitors reveal 
centromeric roles of Aurora B in chromosome movement and spindle checkpoint 
signaling.