PROJECT SUMMARY Astrocytes are essential for the generation, maintenance, and function of neuronal circuits in the central nervous system (CNS). Recent studies from our laboratory revealed that astrocytes exhibit extensive molecular and functional diversity across several regions of the CNS. To understand how this functional diversity is encoded, we identified several transcription factors (TFs) that exhibit region-specific expression. Despite these findings, how TFs contribute to astrocyte physiology and circuit function remain largely undefined. Among the TFs exhibiting region-specific expression, we found that the homeobox TF Nkx6.1 is expressed exclusively in ventral astrocytes of the mature spinal cord. Temporal-conditional deletion of Nkx6.1 from mature astrocytes (Nkx6.1-cKO) resulted in a lower morphological complexity of astrocytes in the ventral spinal cord suggesting that it regulates key properties of these astrocyte populations. Additionally, motor neurons from Nkx6.1-cKO spinal cords demonstrate reduced expression of choline acetyltransferase accompanied by reduced number of VGlut1 synapses. Finally, ChIP-seq analysis shows that Nkx6.1 binds, and possibly regualtes the expression of genes involved in cell morphology, neuron-astrocyte communication, and synaptic maintenance. Together, these results suggest that Nkx6.1 maintains mature astrocyte functions that are essential for the maintenance of synaptic circuits in the spinal cord. Thus, the focus of our proposal is: 1) to determine how Nkx6.1 maintains morphology and physiology of ventral spinal cord astrocytes; 2) to delineate how Nkx6.1-dependent astrocyte functions contribute to the activity of spinal cord motor neurons and motor behaviors, and 3) to define the unique transcriptional mechanism controlled by Nkx6.1 in ventral spinal cord astrocytes.