Hypertrophic chondrocytes and their specific marker, the type X collagen gene (), are critical components of endochondral bone formation during skeletal development. We previously found that Runx2 is an indispensable mouse gene regulator and identified many other transcription factors (TFs) that potentially interact with the 150-bp cis-enhancer. However, the roles of these candidate TFs in expression and chondrocyte hypertrophy have not been elucidated. Here, we focus on 32 candidate TFs recently identified by analyzing the 150-bp enhancer using the transcription factor affinity prediction (TRAP) program. We found that 12 TFs (Hoxa3, Lsx, Evx2, Dlx5, S8, Pax2, Egr2, Mef2a, Barhl2, GKlf, Sox17, and Crx) were significantly upregulated and four TFs (Lhx4, Tbx5, Mef2c, and Hb9) were significantly downregulated in hypertrophic MCT cells, which show upregulation of expression. Most of the differential expression pattern of these TFs conformed with the results obtained from ATDC5 cell model and primary mouse chondrocytes. Notably, was downregulated upon upregulation, overexpression of decreased expression, and knock-down of increased expression in hypertrophic chondrocytes, suggesting that Tbx5 is a negative regulator of . We further generated a stable -overexpressing ATDC5 cell line and transgenic mice driven by -specific enhancers and promoters. overexpression decreased expression in ATDC5 cells cultured as early as day 7 and in limb tissue on post-natal day 1. Slightly weaker alkaline phosphatase staining was also observed in cell culture on day 7 and in limb digits on embryonic day 17.5, indicating mildly delayed ossification. Further characterization of these candidate transcriptional regulators could help identify novel therapeutic targets for skeletal diseases associated with abnormal chondrocyte hypertrophy.