Updated project metadata. Environmental stimuli, including elevated CO2, regulate stomatal development1-3 but the key mechanisms mediating the perception and relay of the CO2 signal to the stomatal development machinery remain elusive. To adapt CO2 intake to water loss, plants regulate the development of stomatal gas exchange pores in the aerial epidermis. Diverse plant species show a decrease in stomatal density in response to the continuing rise of atmospheric CO2 4. To date, one mutant, hic5, defective in cell wall wax biosynthesis, has been identified that exhibits a de-regulation of this CO2-controlled stomatal development response. Here we show that recently isolated Arabidopsis thaliana carbonic anhydrase double mutant plants6 exhibit an inversion in their response to elevated CO2, showing increased stomatal development at elevated CO2 levels. We have characterized the mechanisms mediating this response and demonstrate extracellular signaling in the regulation of CO2-controlled stomatal development by carbonic anhydrases. Transcriptomic RNA-Seq analyses show that the extracellular pro-peptide gene EPF2 7,8, but not EPF1 9, is induced at elevated CO2 in wild type, but not ca1ca4 mutant leaves. Moreover, EPF2 is essential for CO2 control of stomatal development. Using cell wall proteomic and CO2-dependent transcriptome analyses, we have identified a novel, CO2-induced extracellular protease, CRSP (CO2 Response Secreted Protease), as a mediator of CO2 controlled stomatal development. Our results identify mechanisms and genes that function in the repression of stomatal development in leaves during atmospheric CO2 elevation, including the CA1/CA4 carbonic anhydrases and the secreted protease CRSP that cleaves the pro-peptide EPF2, which in turn represses stomatal development.