Cytokinesis in eukaryotes involves specific arrays of microtubules (MTs), which are known as the "central spindle" in animals, the "anaphase spindle" in yeasts, and the "phragmoplast" in plants. Control of these arrays, which are composed mainly of bundled nonkinetochore MTs, is critically important during cytokinesis. In plants, an MAPK cascade stimulates the turnover of phragmoplast MTs, and a crucial aspect of the activation of this cascade is the interaction between the MAPKKK, nucleus- and phragmoplast-localized protein kinase 1 (NPK1) and the NPK1-activating kinesin-like protein 1 (NACK1), a key regulator of plant cytokinesis. However, little is known about the control of this interaction at the molecular level during progression through the M phase. We demonstrated that cyclin-dependent kinases (CDKs) phosphorylate both NPK1 and NACK1 before metaphase in tobacco cells, thereby inhibiting the interaction between these proteins, suggesting that such phosphorylation prevents the transition to cytokinesis. Failure to inactivate CDKs after metaphase prevents dephosphorylation of these two proteins, causing incomplete mitosis. Experiments with Arabidopsis NACK1 (AtNACK1/HINKEL) revealed that phosphorylated NACK1 fails to mediate cytokinesis. Thus, timely and coordinated phosphorylation by CDKs and dephosphorylation of cytokinetic regulators from prophase to anaphase appear to be critical for the appropriate onset and/or progression of cytokinesis.