Abstract: Switching between alternate states of gene transcription is fundamental to a multitude of cellular regulatory pathways, including those that govern differentiation. In spite of the progress in our understanding of such transitions in gene activity, a major unanswered question is how cells regulate the timing of these switches. Here, we have examined the kinetics of a transcriptional switch that accompanies the differentiation of yeast cells of one mating-type into a distinct new cell type. We found that cell type-specific genes silenced by the alpha2 repressor in the starting state are de-repressed to establish the new mating-type-specific gene expression program coincident with the loss of alpha2 from promoters. This rapid de-repression does not require the pre-loading of RNA polymerase II or a preinitiation complex, but instead depends upon the Gcn5 histone acetyltransferase. Surprisingly, Gcn5-dependent acetylation of nucleosomes in the promoters of mating-type-specific genes requires the co-repressor Ssn6-Tup1 even in the repressed state. Gcn5 partially acetylates the amino-terminal tails of histone H3 in repressed promoters, thereby priming them for rapid de-repression upon loss of alpha2. Thus, Ssn6-Tup1 not only efficiently represses these target promoters, it also functions to initiate de-repression by creating a chromatin state poised for rapid activation.