Although replication-coupled chromatin assembly is known to be important for the maintenance of patterns of gene expression through sequential cell divisions, the role of replication-coupled chromatin assembly in controlling cell differentiation during animal development remains largely unexplored. Here we report that the CAF-1 protein complex, an evolutionarily conserved histone chaperone that deposits histone H3-H4 proteins onto replicating DNA, is required to generate a bilateral asymmetry in the C. elegans nervous system. A mutation in 1 of 24 C. elegans histone H3 genes specifically eliminates this aspect of neuronal asymmetry by causing a defect in the formation of a histone H3-H4 tetramer and the consequent inhibition of CAF-1-mediated nucleosome formation. Our results reveal that replication-coupled nucleosome assembly is necessary to generate a bilateral asymmetry in C. elegans neuroanatomy and suggest that left-right asymmetric epigenetic regulation can establish bilateral asymmetry in the nervous system. Display omitted ► CAF-1 is required for a neuronal bilateral asymmetry in C. elegans ► A mutation in 1 of 24 histone H3 genes specifically eliminates this asymmetry ► Mutant histone H3 proteins inhibit CAF-1-mediated nucleosome formation ► A defect in the formation of H3-H4 tetramers causes loss of bilateral asymmetry Mutation of a histone H3 gene in C. elegans disrupts the left-right asymmetry of a pair of neurons, revealing a role for asymmetric nucleosome assembly in development.