Heat shock factors (HSFs) are best known as transcriptional regulators of genes encoding Hsps and other stress proteins. The HSF family in vertebrates, HSF1-4, are also important for normal development and lifespan-regulating pathways. Distinct HSFs possess unique and overlapping functions, exhibit tissue-specific expression patterns, undergo multiple post-translational modifications, such as acetylation, phosphorylation, sumoylation and ubiquitylation, and interact with diverse protein partners. Our studies are focused on mammalian HSF1 and HSF2, between which we have demonstrated a physical and functional interplay both in cell stress and developmental processes, such as spermatogenesis. Recent findings have expanded the repertoire of HSF targets well beyond the Hsp genes, which has considerably changed the view of HSFs solely regulating stress-inducible gene expression. Simultaneous disruption of Hsf1 and Hsf2 causes a severe testis phenotype associated with male sterility, suggesting that transcriptional activity of both factors is required for normal spermatogenesis. A large set of novel target genes for HSF1 and HSF2 was discovered by chromatin immunoprecipitation on promoter microarray (ChIP-chip) screens in mouse testis. Of particular interest are the sex chromosomal multi-copy genes that are important for sperm differentiation and sperm head development. Given that the sex chromatin mostly remains silent after meiosis, HSF1 and HSF2 are unique in contributing to the transcriptional regulation of sex-chromosomal genes during post-meiotic repression. The regulation of differential expression profiles of HSF1 and HSF2 during spermatogenesis will be discussed.