The initiation of stomatal lineage and subsequent asymmetric divisions in Arabidopsis require the activity of the basic helix-loop-helix transcription factor SPEECHLESS (SPCH). It has been shown that SPCH controls entry into the stomatal lineage as a substrate either of the MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) cascade or GSK3-like kinase BRASSINOSTEROID INSENSITIVE 2 (BIN2). Here we show that three serine residues of SPCH appear to be the primary phosphorylation targets of Cyclin-Dependent Kinases A;1 (CDKA;1) in vitro, and among them Serine 186 plays a crucial role in stomatal formation. Expression of an SPCH construct harboring a mutation that results in phosphorylation deficiencies on Serine 186 residue failed to rescue stomatal defects in spch null mutants. Expression of a phosphorylation-mimic mutant SPCH(S186D) complemented stomatal production defects in the transgenic lines harboring the targeted expression of dominant-negative CDKA;1.N146. Therefore, in addition to MAPK- and BIN2-mediated phosphorylation on SPCH, phosphorylation at Serine 186 is positively required for SPCH function in regulating stomatal development.