The switch from cellular proliferation to differentiation occurs to a large extent through specific programs of gene expression. In fission yeast, the master regulator of sexual differentiation, ste11, is induced by environmental conditions leading to mating and meiosis. We show that phosphorylation of serine 2 (S2P) in the C-terminal domain of the largest subunit of the RNA polymerase II (PolII) enzyme by the Lsk1 cyclin-dependent kinase has only a minor impact on global gene expression during vegetative growth but is critical for the induction of ste11 transcription during sexual differentiation. The recruitment of the Lsk1 kinase initiates in the vicinity of the transcription start site of ste11, resulting in a marked increase of S2P on the ste11 unit, including an extended 5' untranslated region (5'UTR). This pattern contrasts with the classical gradient of S2P toward the 3' region. In the absence of S2P, both PolII occupancy at the ste11 locus and ste11 expression are impaired. This results in sterility, which is rescued by expression of the ste11 coding sequence from the adh1 promoter. Thus, the S2P polymerase plays a specific, regulatory role in cell differentiation through the induction of ste11.