Astrocytes, a type of glial cells, have multiple roles in regulating neuronal development and homeostasis. In addition to the typical mammalian astrocytes, in the primate cortex interlaminar astrocytes are located in the superficial layer and project long processes traversing multiple layers of the cerebral cortex. Previously, we described a human stem cell based chimeric mouse model where interlaminar astrocytes develop. Here, we utilized this model to study the calcium signaling properties of interlaminar astrocytes. To determine how interlaminar astrocytes could contribute to neurodevelopmental disorders, we generated a chimeric mouse model for Fragile X syndrome. We report that FXS interlaminar astrocytes exhibit hyperexcitable calcium signaling and cause an increase in dendritic spine dynamics.