Stomata are pores in the plant epidermis that control carbon dioxide uptake and water loss. They are major regulators of global carbon and water cycles . Several signaling components that regulate stomatal development have been characterized. These include a putative secretory peptide EPF1, LRR receptor components TMM and ER, and a peptidase SDD1 [2-4]. We have identified EPF2, a peptide related to EPF1 that is expressed in proliferating cells of the stomatal lineage, known as meristemoids, and in guard mother cells, the progenitors of stomata. EPF2 expression during leaf development affects stomatal density on the mature leaf. In the absence of EPF2, excessive numbers of cells enter the stomatal lineage and produce numerous small epidermal cells that express stomatal lineage reporter genes, whereas plants overexpressing EPF2 produce virtually no stomata. Results from genetic experiments indicate that EPF2 regulates a different aspect of stomatal development to EPF1 and are consistent with EPF2 acting in a pathway to regulate stomatal density that involves ER and TMM, but not SDD1. We propose that EPF2 is expressed earlier in leaf development than EPF1 and is involved in determining the number of cells that enter, and remain in, the stomatal lineage.