Significance We have shown that the immunoglobulin heavy chain V region mutation in human B cells is regulated by HIRA, which is a chaperone for H3.3, and found that H3.3 plays a role in V region mutation in part through its H3.3K36me3 modification. The absence of HIRA is associated with a change in V region chromatin accessibility. HIRA depletion and expression of the H3.3G34V mutant also decreased the recruitment of several transcriptional elongation factors, including ZMYND11, which had not previously been shown to be involved in V region mutation. These and other findings extend our knowledge of the role of chromatin structure and of transcription in regulating V region hypermutation.