The study investigates the regulatory mechanisms underlying heat stress (HS) tolerance in maize (Zea mays). Using transcriptome data from short- and long-term heat treatments, the authors identified the transcription factor ZmHSF20, a member of the class B2a heat shock factor (HSF) family. ZmHSF20 was found to bind to the promoters of Cellulose synthase A2 (ZmCesA2) and three class A Hsf genes, including ZmHsf4, repressing their transcription. Overexpression of ZmCesA2 and ZmHsf4 improved HS response, with ZmHsf4 directly activating ZmCesA2 transcription. ZmHSF20 inhibited cellulose accumulation and repressed cell wall-related gene expression, leading to reduced thermotolerance in ZmHsf20 mutant seedlings. The double mutant ZmHsf20 ZmHsf4 exhibited decreased thermotolerance, placing ZmHsf4 downstream of ZmHsf20. The authors propose an expanded model of the HS response in maize, where ZmHSF20 negatively regulates ZmHsf4 and ZmCesA2 to balance seedling growth and defense. This study highlights the role of ZmHSF20 in modulating HS tolerance through its interaction with ZmHsf4 and ZmCesA2, providing insights into the genetic basis of HS tolerance in maize.The study investigates the regulatory mechanisms underlying heat stress (HS) tolerance in maize (Zea mays). Using transcriptome data from short- and long-term heat treatments, the authors identified the transcription factor ZmHSF20, a member of the class B2a heat shock factor (HSF) family. ZmHSF20 was found to bind to the promoters of Cellulose synthase A2 (ZmCesA2) and three class A Hsf genes, including ZmHsf4, repressing their transcription. Overexpression of ZmCesA2 and ZmHsf4 improved HS response, with ZmHsf4 directly activating ZmCesA2 transcription. ZmHSF20 inhibited cellulose accumulation and repressed cell wall-related gene expression, leading to reduced thermotolerance in ZmHsf20 mutant seedlings. The double mutant ZmHsf20 ZmHsf4 exhibited decreased thermotolerance, placing ZmHsf4 downstream of ZmHsf20. The authors propose an expanded model of the HS response in maize, where ZmHSF20 negatively regulates ZmHsf4 and ZmCesA2 to balance seedling growth and defense. This study highlights the role of ZmHSF20 in modulating HS tolerance through its interaction with ZmHsf4 and ZmCesA2, providing insights into the genetic basis of HS tolerance in maize.