Cell-to-cell signalling is a major mechanism controlling plant morphogenesis. Transport of signalling molecules through plasmodesmata is one way in which plants promote or restrict intercellular signalling over short distances. Plasmodesmata are membrane-lined pores between cells that regulate the intercellular flow of signalling molecules through changes in their size, creating symplasmic fields of connected cells. Here we examine the role of plasmodesmata and symplasmic communication in the establishment of plant cell totipotency, using somatic embryo induction from Arabidopsis explants as a model system. Cell-to-cell communication was evaluated using fluorescent tracers, supplemented with histological and ultrastructural analysis, and correlated with expression of a WOX2 embryo reporter. We showed that embryogenic cells are isolated symplasmically from non-embryogenic cells regardless of the explant type (immature zygotic embryos or seedlings) and inducer system (2,4-dichlorophenoxyacetic acid or the BABY BOOM (BBM) transcription factor), but that the symplasmic domains in different explants differ with respect to the maximum size of molecule capable of moving through the plasmodesmata. Callose deposition in plasmodesmata preceded WOX2 expression in future sites of somatic embryo development, but later was greatly reduced in WOX2-expressing domains. Callose deposition was also associated with a decrease DR5 auxin response in embryogenic tissue. Treatment of explants with the callose biosynthesis inhibitor 2-deoxy-D-glucose supressed somatic embryo formation in all three systems studied, and also blocked the observed decrease in DR5 expression. Together these data suggest that callose deposition at plasmodesmata is required for symplasmic isolation and establishment of cell totipotency in Arabidopsis.