AFF4 is a component of super elongation complex (SECs) and functions as a scaffold protein to bridge the transcription elongation factors. It is associated with leukemia, HIV transcription, and head neck cancer. However, its role in odontogenic differentiation of dental pulp cells (DPCs) is unclear. Here, we show the expression of AFF4 is increased during odontogenesis. Depletion of AFF4 in human DPCs leads to a decrease of alkaline phosphatase (ALP) activity, calcium mineralization and odontogenic-related genes expression. On the contrary, Lentivirus-mediated overexpression of AFF4 induces the odontogenic potential of DPCs. Mechanistically, we found AFF4 regulates the transcription of NFIC, a key factor for tooth root formation. Overexpression of NFIC successfully rescues the restricted differentiation of AFF4-depleted cells. Our data demonstrate that AFF4 serves as a previously unknown regulator of odontogenesis. (C) 2020 Published by Elsevier Inc.