RNA polymerase III transcribes small untranslated RNAs that fulfill essential cellular functions in regulating transcription, RNA processing, translation and protein translocation. RNA polymerase III transcription activity is tightly regulated during the cell cycle and coupled to growth control mechanisms. Furthermore, there are reports of changes in RNA polymerase III transcription activity during cellular differentiation, including the discovery of a novel isoform of human RNA polymerase III that has been shown to be specifically expressed in undifferentiated human H1 embryonic stem cells. Here, we review major regulatory mechanisms of RNA polymerase III transcription during the cell cycle, cell growth and cell differentiation.