In plants, stomata on the aerial epidermis play critical roles in various biological processes, including gas exchange, photosynthesis, transpiration, and immunity. Stomatal development is negatively and positively controlled by the mitogen-activated protein kinase (MAPK) cascade and nitric oxide (NO), respectively. However, the regulatory scheme of stomatal development by these signaling pathways remains elusive. Here, we show that NO-controlled stomatal development in Arabidopsis is genetically dependent on MPK3 and MPK6. Moreover, NO-controlled S-nitrosylation of MPK6 at cysteine (Cys)-201 inhibits its phosphorylation, resulting in the stabilization ofSPEECHLESS (SPCH), a master regulator of stomatal lineage initiation, thereby promoting stomatal development. AnMPK6C201S mutation confers NO insensitivity during stomatal development and stress responses. We propose that NO positively controls stomatal development and stress responses by inhibiting the MPK6 activity via S-nitrosylation, thus identifying a mechanism linking the coupled NO-MAPK signaling to specific biological outputs.