Cell-to-cell communication coordinates the behavior of individual cells to establish organ patterning and development. Although mobile signals are known to be important in lateral root development, the role of plasmodesmata (PD)-mediated transport in this process has not been investigated. Here, we show that changes in symplastic connectivity accompany and regulate lateral root organogenesis in Arabidopsis. This connectivity is dependent upon callose deposition around PD affecting molecular flux through the channel. Two plasmodesmal-localized ß-1,3 glucanases (PdBGs) were identified that regulate callose accumulation and the number and distribution of lateral roots. The fundamental role of PD-associated callose in this process was illustrated by the induction of similar phenotypes in lines with altered callose turnover. Our results show that regulation of callose and cell-to-cell connectivity is critical in determining the pattern of lateral root formation, which influences root architecture and optimal plant performance.