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In eukaryotes, MEDIATOR is a conserved multi-subunit complex that links transcription factors and RNA polymerase Ⅱ and that thereby facilitates transcriptional initiation. Although the composi-tion of MEDIATOR has been well studied in yeast and mammals, relatively little is known about the composition of MEDIATOR in plants. By affinity purification followed by mass spectrometry, we identified 28 conserved MEDIATOR subunits in Arabidopsis thaliana, including putative MEDI-ATOR subunits that were not previously validated. Our results indicated that MED34, MED35, MED36, and MED37 are not Arabidopsis MEDI-ATOR subunits, as previously proposed. Our re-sults also revealed that two homologous CBP/p300 histone acetyltransferases, HAC1 and HAC5 (HAC1/5) are in fact plant-specific MEDIATOR subunits. The MEDIATOR subunits MED8 and MED25 (MED8/25) are partially responsible for the association of MEDIATOR with HAC1/5, MED8/25 and HAC1/5 co-regulate gene expression and thereby affect flowering time and floral develop-ment. Our in vitro observations indicated that MED8 and HAC1 form liquid-like droplets by phase separation, and our in vivo observations indicated that these droplets co-localize in the nuclear bodies at a subset of nuclei. The formation of liquid-like droplets is required for MED8 to in-teract with RNA polymerase Ⅱ. In summary, we have identified all of the components of Arabi-dopsis MEDIATOR and revealed the mechanism underlying the link of histone acetylation and transcriptional regulation.