Signaling mediates cellular responses to extracellular stimuli. The c-Jun NH(2)-terminal kinase (JNK) pathway exemplifies one subgroup of the mitogen-activated protein (MAP) kinases, which, besides having established functions in stress response, also contribute to development by an unknown mechanism. We show by genome-wide location analysis that JNK binds to a large set of active promoters during the differentiation of stem cells into neurons. JNK-bound promoters are enriched with binding motifs for the transcription factor NF-Y but not for AP-1. NF-Y occupies these predicted sites, and overexpression of dominant-negative NF-YA reduces the JNK presence on chromatin. We find that histone H3 Ser10 (H3S10) is a substrate for JNK, and JNK-bound promoters are enriched for H3S10 phosphorylation. Inhibition of JNK signaling in post-mitotic neurons reduces phosphorylation at H3S10 and the expression of target genes. These results establish MAP kinase binding and function on chromatin at a novel class of target genes during stem cell differentiation.