In Arabidopsis thaliana L., stomata are produced through a series of divisions including asymmetric and symmetric divisions. Asymmetric entry division of meristemoid mother cell produces two daughter cells, the smaller meristemoid and the larger sister cell, a stomatal lineage ground cell (SLGC). Stomatal lineage ground cells can differentiate into epidermal pavement cells but have the potential to divide asymmetrically, spacing divisions, to create satellite meristemoids. Peptide ligands and TOO MANY MOUTHS (TMM) and ERECTA family receptors regulate the initiation of stomatal lineages, activity, and orientation of spacing divisions. Here, we reported that a natural mutant 28y displayed an increased stomatal density and index. Using map-based cloning, we identified mutation in ARGONAUTE1 (AGO1) as the cause of 28yphenotypes. Time-lapse tracing of stomatal lineage cells reveals that stomatal overproduction in 28y is caused by the excessive asymmetric spacing division of SLGCs. Further genetic results demonstrated that AGO1 acts downstream of TMM and negatively regulates the SPCHtranscripts, but in a brassinosteroid-independent manner. Upregulation of AGAMOUS-LIKE16 (AGL16) in 28y mutants suggests that AGO1 is required to restrict AGL16-mediated stomatal spacing divisions, an miRNA pathway in addition to ligand-receptor signaling modules.