Mediator, a transcriptional co-activator complex, is recruited to enhancers by DNA-binding activators via its tail module, and it interacts with RNA polymerase (Pol) II as part of the preinitiation complex via its head module. Although Mediator has been described as a general transcription factor, there are conflicting evidences for whether it is essential for Pol II transcription in vivo. Here, we perform a comprehensive analysis of this issue by using the anchor-away approach to rapidly deplete individual or multiple Mediator subunits from the nucleus. Depletion of individual subunits, even those essential for cell growth, cause a general but modest decrease in Pol II transcription. Transcriptional effects are stronger on SAGA-dependent genes as opposed to TFIID-dependent genes. Thus, Mediator modules that associate either with the enhancer or with the core promoter confer partial transcriptional activity. In contrast, simultaneous depletion of all Mediator modules causes a drastic decrease in Pol II transcription that is roughly comparable to the effect observed upon anchor-away-mediated depletion of Pol II or TBP. These results provide strong evidence that Mediator is essential for Pol II transcription in vivo, but that it is not a required component of the preinitiation