Abstract FLOWERING LOCUS T (FT) is the major regulatory component controlling photoperiodic floral transition. It is expressed in guard cells and affects blue light-induced stomatal opening induced by the blue-light receptor phototropins phot1 and phot2. Roles for other flowering regulators in stomatal opening have yet to be determined. We show in Arabidopsis (Arabidopsis thaliana) that TWIN SISTER OF FT (TSF), CONSTANS (CO), and GIGANTEA (GI) provide a positive effect on stomatal opening. TSF, which is the closest homolog of FT, was transcribed in guard cells, and light-induced stomatal opening was repressed in tsf-1, a T-DNA insertion mutant of TSF. Overexpression of TSF in a phot1 phot2 mutant background gave a constitutive open-stomata phenotype. Then, we examined whether CO and GI, which are upstream regulators of FT and TSF in photoperiodic flowering, are involved in stomatal opening. Similar to TSF, light-induced stomatal opening was suppressed in the GI and CO mutants gi-1 and co-1. A constitutive open-stomata phenotype was observed in GI and CO overexpressors with accompanying changes in the transcription of both FT and TSF. In photoperiodic flowering, photoperiod is sensed by photoreceptors such as the cryptochromes cry1 and cry2. We examined stomatal phenotypes in a cry1 cry2 mutant and in CRY2 overexpressors. Light-induced stomatal opening was suppressed in cry1 cry2, and the transcription of FT and TSF was down-regulated. In contrast, the stomata in CRY2 overexpressors opened even in the dark, and FT and TSF transcription was up-regulated. We conclude that the photoperiodic flowering components TSF, GI, and CO positively affect stomatal opening.