The functions of cellular prion protein (PrPC) are under intense debate and PrPC loss of function has been implicated in the pathology of prion diseases. Neuronal PrPC engagement with stress-inducible protein-1 and laminin (LN) plays a key role in cell survival and differentiation. The present study evaluated whether PrPC expression in astrocytes modulates neuron-glia cross-talk that underlies neuronal survival and differentiation. Astrocytes from wild-type mice promoted a higher level neuritogenesis than astrocytes obtained from PrPC-null animals. Remarkably, neuritogenesis was greatly diminished in co-cultures combining PrPC-null astrocytes and neurons. LN secreted and deposited at the extracellular matrix by wild-type astrocytes presented a fibrillary pattern and was permissive for neuritogenesis. Conversely, LN coming from PrPC-null astrocytes displayed a punctate distribution, and did not support neuronal differentiation. Additionally, secreted soluble factors from PrPC-null astrocytes promoted lower levels of neuronal survival than those secreted by wild-type astrocytes. PrPC and stress-inducible protein-1 were characterized as soluble molecules secreted by astrocytes which participate in neuronal survival. Taken together, these data indicate that PrPC expression in astrocytes is critical for sustaining cell-to-cell interactions, the organization of the extracellular matrix, and the secretion of soluble factors, all of which are essential events for neuronal differentiation and survival.