The dehydration-responsive element binding protein (DREB)/C-repeat binding factor (CBF) family are the classical transcriptional regulators involved in plant responses to drought, salt and cold stress. Recently it was demonstrated that DREB2A is induced by heat stress (hs) and is a regulator of the hs response of Arabidopsis. Here we provide molecular insights into the regulation and function of hs transcription factor HsfA3. Among the 21 members of the Arabidopsis Hsf family, HsfA3 is the only Hsf that is transcriptionally induced during hs by DREB2A, and HsfA3 in turn regulates the expression of Hsp-encoding genes. This transcription factor cascade was reconstructed in transient GUS reporter assays in mesophyll protoplasts by showing that DREB2A could activate the HsfA3 promoter, whereas HsfA3 in turn was shown to be a potent activator on the promoters of Hsp genes. Direct binding to the corresponding promoters was demonstrated by electrophoretic mobility shift assays, and the involvement of HsfA3 in the hs response in vivo was shown directly by observation of reduced thermotolerance in HsfA3 mutant lines. Altogether these data demonstrate that HsfA3 is transcriptionally controlled by DREB2A and important for the establishment of thermotolerance.