Updated project metadata. The Greatwall/Mastl kinase is an essential gene, which regulates the phosphatase activity that counteracts the phosphorylation of Cdk1 substrates. Although Mastl-/- MEFs can enter mitosis, they are unable to complete mitosis due to chromosome segregation defects. Here, we show that Mastl is essential for robust spindle assembly checkpoint (SAC) maintenance. Mastl-/- MEFs display reduced phosphorylation and kinase activity of MPS1, which causes mislocalization of MPS1, Mad1, and Aurora B from the kinetochore and the inner centromere regions. This results in premature inactivation of SAC signalling and early anaphase onset without correction of chromosome-spindle attachment mistakes. Treatment of the knockout cells with protein phosphatase II inhibitor okadaic acid (OA) rescued decreased MPS1 activity, mislocalization of Aurora B, Mad1, MPS1, and premature mitotic slippage. Our data demonstrates how regulation of PP2A activity by Mastl kinase prevents premature SAC silencing as a result of controlling the phosphorylation status and activity of MPS1.