ABSTRACTInitiation of flowering is a crucial developmental event that requires both internal and environmental signals to determine when floral transition should occur to maximize reproductive success. Ambient temperature is one of the key environmental signals that highly influence flowering time, not only seasonally but also in the context of drastic temperature fluctuation due to global warming. Molecular mechanisms of how high or low constant temperatures affect the flowering time have been largely characterized in the model plantArabidopsis thaliana; however, the effect of natural daily variable temperature outside laboratories is only partly explored. Several groups of flowering genes have been shown to play important roles in temperature responses, including two temperature-responsive transcription factors (TFs), namely PHYTOCHROME INTERACTING FACTOR 4 (PIF4) and FLOWERING LOCUS C (FLC), that act antagonistically to regulate flowering time by activating or repressing floral integratorFLOWERING LOCUS T (FT). In this study, we have demonstrated that the daily variable temperature (VAR) causes early flowering in both natural accessions Col-0, C24 and their late flowering hybrid C24xCol, which carries both functional floral repressor FLC and its activator FRIGIDA (FRI), as compared to a constant temperature (CON). The loss-of-function mutation ofPIF4exhibits later flowering in VAR, suggesting that PIF4 at least in part, contributes to acceleration of flowering in response to the daily variable temperature. We find that VAR increasesPIF4transcription at the end of the day when temperature peaks at 32 °C. TheFTtranscription is also elevated in VAR, as compared to CON, in agreement with earlier flowering observed in VAR. In addition, VAR causes a decrease inFLCtranscription in 4-week-old plants, and we further show that overexpression ofPIF4can reduceFLCtranscription, suggesting that PIF4 might also regulateFTindirectly through the repression ofFLC. To further conceptualize an overall model of gene regulatory mechanisms involvingPIF4andFLCin controlling flowering in response to temperature changes, we construct a co-expression – transcriptional regulatory network by combining publicly available transcriptomic data and gene regulatory interactions of our flowering genes of interest and their partners. The network model reveals the conserved and tissue-specific regulatory functions of 62 flowering-time-relating genes, namelyPIF4,PIF5,FLC,ELF3and their immediate neighboring genes, which can be useful for confirming and predicting the functions and regulatory interactions between the key flowering genes.