Pure titanium surface is strongly bound to the bone when embedded as a dental implant, with high biocompatibility to the soft tissue around the implant. However, the mechanical strength of Ti-6Al-4V alloy is superior to that of pure titanium. Titanium alloy products account for about 40% of all implant products in Japan. Of the composition elements of Ti-6Al-4V alloy, vanadium has a higher toxicity than the others. Ti-6Al-4V alloys are employed in various titanium alloy implant products. In the present study, cell differentiation and proliferation levels were examined with a polished Ti-6Al-4V alloy in three-dimensional ES cell culture on collagen, demonstrating no developmental toxicity of Ti-6Al-4V alloy. However, Ti-6Al-4V alloy powder corroded with a hydrofluoric acid solution had effects on the differentiation and proliferation of ES cells.