Tooth development is regulated by reciprocal interaction between epithelial and mesenchymal cells in direct and indirect manner. Amelogenesis imperfecta (AI) is inherited enamel defects caused by alteration of gene regulations involved in enamel formation and maturation. We have identified that Specificity protein 6 (SP6), a transcription factor, is one of the AI-causative genes using spontaneous mutant AI-rat (Muto et al. Orphanet J Rare Dis. 2012), which is followed by establishment of AI-derived dental epithelial cells (ARE- B30) (Adiningrat et al. Oral Dis. 2016). To further analyze the characteristics, we compared the amelogenesis-related gene expression between ARE-B30 and its wild type control, G5 cells, using 3D culture systems; collagen membrane (CM-6), or co-cultured with RPC-C2A (rat pulp cells) on CM-6 in separated and mixed medium, as well as conventional plastic culture. Reciprocal expression of Bmp2 and Fst, and enhancement of Amtn were observed in G5 by 3D co-culture system. Furthermore, G5 expressed Klk4, but not Mmp20 among all culture system. In contrast, no expression of Amtn, Klk4 and Mmp20 were observed in ARE-B30. Importantly, 3D culture system could demonstrate the distinct effects of ECM and mesenchyme on gene expression in ARE-B30 compared to G5. From this study, we found AI-derived dental epithelial cells have unique responsiveness to the in vivo mimicking culture condition, possibly reflecting the different or aberrant cell differentiation compared to wild type control.