Plasmodesmata are nanoscale cell wall channels connecting neighboring cells in plants. Intercellular trafficking of moleculesvia plasmodesmata plays important roles in various developmental processes and stress responses. The turnover of callose, a β-1,3-glucan polysaccharide depositing in the cell wall around plasmodesmata, controls the plasmodesmal permeability and symplasmic transport. Here, we describe a protocol for the spatiotemporally controlled induction of callose synthesis and plasmodesmata closure using the cals3m system. In this system, cals3m, a mutant CALLOSE SYNTHASE 3 (CALS3) gene, is driven by inducible tissue-specific promoters of interest. After appropriate induction by 17-β-estradiol, callose is overproduced within the corresponding specific domains, resulting in temporal closure of plasmodesmata at the cell-cell interfaces. This approach can be used to validate and dissect the function of plasmodesmata-mediated symplasmic communications.