Stage-specific expression of ameloblast-specific genes is controlled by differential expression of transcription factors. In addition, ameloblasts follow daily rhythms in their main activities (i.e. enamel protein secretion and enamel mineralization). This time-related control is orchestrated by oscillations of clock proteins involved in the regulation of circadian rhythms. Our aim was to identify the potential links between daily rhythms and developmental controls of ameloblast differentiation. The effects of the transcription factors distal-less homeobox 3 (Dlx3) and runt-related transcription factor 2 (Runx2), and the clock gene nuclear receptor subfamily 1, group D, member 1 (Nr1d1), on secretory and maturation ameloblasts [using stage-specific markers amelogenin (Amelx), enamelin (Enam), and kallikrein-related peptidase 4 (Klk4)] were evaluated in the HAT-7 ameloblast cell line. Amelx and Enam steady-state mRNA expression levels were down-regulated in Runx2 over-expressing cells and up-regulated in Dlx3 over-expressing cells. In contrast, Klk4 mRNA was up-regulated by both Dlx3 and Runx2. Furthermore, a temporal and spatial relationship between clock genes and ameloblast differentiation markers was detected. Of interest, clock genes not only affected rhythmic expression of ameloblast-specific genes but also influenced the expression of Runx2. Multiscale mathematical modeling is being explored to further understand the temporal and developmental controls of ameloblast differentiation. Our study provides novel insights into the regulatory mechanisms sustaining ameloblast differentiation.