Regulation of gene expression is fundamental in all living organisms and is facilitated by transcription factors, the single largest group of proteins in humans. For cell- and stimulus-specific gene regulation, strict control of the transcription factors themselves is crucial. Heat shock factors are a family of transcription factors best known as master regulators of induced gene expression during the heat shock response. This evolutionary conserved cellular stress response is characterized by massive production of heat shock proteins, which function as cytoprotective molecular chaperones against various proteotoxic stresses. In addition to promoting cell survival under stressful conditions, heat shock factors are involved in the regulation of life span and progression of cancer and they are also important for developmental processes such as gametogenesis, neurogenesis and maintenance of sensory organs. Here, we review the regulatory mechanisms steering the activities of the mammalian heat shock factors 1–4.