The signaling pathways orchestrating both the evolution and development of language in the human brain remain unknown. To date, the transcription factor FOXP2 is the only gene implicated in Mendelian forms of human speech and language dysfunction1,2. It has been proposed, that the amino acid composition in the human variant of FOXP2 has undergone accelerated evolution, and this change occurred around the time of language emergence in humans3,4. However, this remains controversial, and whether the acquisition of these amino acids in human FOXP2 has any functional consequence in human neurons remains untested. Here, we demonstrate that these two amino acids confer new functionality in terms of differential transcriptional regulation, and extend these observations to in vivo brain, showing that several of the differential FOXP2 targets significantly overlap with genes different between human and chimpanzee brain. We also identify novel relationships among the differentially expressed genes with additional critical regulators of neuronal development. These data provide support for the functional relevance of changes that occur on the human lineage by showing that the two amino acids unique to human FOXP2 can lead to significant differences in gene expression patterns across brain evolution, with direct consequences for human brain development and disease. Since FOXP2 has an important role in the use of language in humans, the identified targets may have a critical function in the development and evolution of language circuitry in humans.