Stem cell specification in multicellular organisms relies on the precise spatiotemporal control of RNA polymerase II (Pol II)-dependent gene transcription, in which the evolutionarily conserved Mediator coactivator complex plays an essential role. In Arabidopsis thaliana, SHORTROOT (SHR) and SCARECROW (SCR) orchestrate a transcriptional program that determines the fate and asymmetric divisions of stem cells generating the root ground tissue. The mechanism by which SHR/SCR relays context-specific regulatory signals to the Pol II general transcription machinery is unknown. Here, we report the role of Mediator in controlling the spatiotemporal transcriptional output of SHR/SCR during asymmetric division of stem cells and ground tissue patterning. The Mediator subunit MED31 interacted with SCR but not SHR. Reduction of MED31 disrupted the spatiotemporal activation of CYCLIND6;1 (CYCD6;1), leading to defective asymmetric division of stem cells generating ground tissue. MED31 was recruited to the promoter of CYCD6;1 in a SCR-dependent manner. MED31 was involved in the formation of a dynamic MED31/SCR/SHR ternary complex through the interface protein SCR. We demonstrate that the relative protein abundance of MED31 and SHR in different cell types regulates the dynamic formation of the ternary complex, which provides a tunable switch to strictly control the spatiotemporal transcriptional output. This study provides valuable clues to understand the mechanism by which master transcriptional regulators control organ patterning.