Hox genes are crucial in determining segmentation identity in developing embryos, which ultimately sets an anteroposterior body axis. Over a century of research has discovered the fundamentals of the Hox gene and protein function in animal development and diseases. However, there are still fundamental questions about the specificity of HOX function. This chapter discusses many unknowns about Hox genes and how modern technologies can overcome technical limitations. We have also addressed these questions to gain a better understanding of the roles of these genes.