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 regulated 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-modulated stomatal development is genetically dependent on MPK3 and MPK6. Moreover, NO-mediated S-nitrosylation of MPK6 at Cys-201 inhibits its phosphorylation, resulting in the stabilization of SPCH, a master regulator of stomatal lineage initiation, thereby promoting stomatal development. Remarkably, a MPK6C201S mutation confers NO insensitivity during stomatal development and stress responses. We propose that NO positively regulates stomatal development and stress responses by the S-nitrosylation-mediated inhibition of the MPK6 activity, thus identifying a mechanism linking the coupled NO-MAPK signaling to specific biological outputs.