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Stomatal development is mediated by EPIDERMAL PATTERNING FACTORs (EPFs), a family of secreted peptides including STOMAGEN/EPFL9 in Arabidopsis. To clarify the functional role of STOMAGEN orthologues in maize (Zea mays), we generated a double knockout mutant of ZmSTOMAGEN1 and ZmSTOMAGEN2 using CRISPR/Cas9 system. Comprehensive phenotypic analysis revealed that the zmstomagen1/2 mutant exhibited severe stomatal development defects, including complete absence of stomata between epidermal cells in stomatal lineage files and abnormal stomatal complexes with small lobed cells. These aberrant cells likely arose from failed asymmetric divisions of guard mother cells, ultimately preventing the formation of functional stomatal complexes. A double knockout of ZmSTOMAGEN1/2 reduced the expression of SPEECHLESS1 (SPCH1), MUTE, SCREAM2 (SCRM2), and STOMATAL DENSITY AND DISTRIBUTION1 (SDD1), impairing stomatal initiation and cell fate transition in early stomatal lineage cells. The mutant displayed a lower stomatal density and index, leading to reduced net photosynthetic rate, transpiration rate, and stomatal conductance but increased water-use efficiency (WUE). Compared to the wild-type plants (HiII-A × HiII-B), the zmstomagen1/2 mutant exhibited significant alterations in phytohormone homeostasis. These included brassinosteroid metabolite imbalance (increased typhasterol, decreased castasterone) and differential gibberellin regulation (elevated GA4, reduced GA1). These hormonal perturbations suggest that impaired stomatal morphogenesis in zmstomagen1/2 mutants result from disrupted crosstalk between multiple hormonals signaling networks. Our findings reveal a crucial role for ZmSTOMAGEN1/2 in regulating cell fate decisions within the stomatal lineage and provide a potential strategy for enhancing WUE in maize by manipulating ZmSTOMAGEN1/2 expression.气孔发育受表皮模式因子（EPIDERMAL PATTERNING FACTORs，EPFs）家族介导，该家族包含拟南芥中的气孔蛋白/EPFL9（STOMAGEN/EPFL9）。为明确玉米（Zea mays）中STOMAGEN同源基因的功能，本研究利用CRISPR/Cas9系统构建了ZmSTOMAGEN1与ZmSTOMAGEN2的双敲除突变体。综合表型分析结果显示，zmstomagen1/2突变体表现出严重的气孔发育缺陷，具体包括气孔谱系细胞列的表皮细胞间完全缺失气孔，以及伴随小型裂叶细胞的异常气孔复合体。这类异常细胞大概率源于保卫母细胞的不对称分裂失败，最终阻碍了功能性气孔复合体的形成。ZmSTOMAGEN1/2双敲除会降低SPEECHLESS1（SPCH1）、MUTE、SCREAM2（SCRM2）以及STOMATAL DENSITY AND DISTRIBUTION1（SDD1）的表达水平，损伤早期气孔谱系细胞的气孔起始与细胞命运转换过程。该突变体的气孔密度与气孔指数均有所降低，进而导致净光合速率、蒸腾速率及气孔导度下降，但水分利用效率（WUE）有所提升。与野生型植株（HiII-A × HiII-B）相比，zmstomagen1/2突变体的植物激素稳态发生显著改变，包括油菜素内酯代谢失衡（梯斯甾醇含量升高、油菜甾酮含量降低）以及赤霉素调控异常（赤霉素4（GA4）水平上升、赤霉素1（GA1）水平下降）。上述激素扰动表明，zmstomagen1/2突变体的气孔形态建成受损，源于多条激素信号网络间的串扰被破坏。本研究揭示了ZmSTOMAGEN1/2在调控气孔谱系细胞命运决定中的关键作用，并为通过调控ZmSTOMAGEN1/2表达提升玉米水分利用效率提供了潜在策略。