Fabrication of engineered skin substitutes provides an alternative approach for the treatment of full-thickness burns and other skin injuries. Improving the functionality of current skin substitute models requires incorporation of skin appendages, including hair follicles, sebaceous glands, and sweat glands. In this chapter, methods for generating skin substitutes incorporating chimeric hair follicles are described. Isolation of human keratinocytes, human fibroblasts, and murine dermal papilla cells is first outlined. These cell types are then combined with collagen-glycosaminoglycan (GAG) scaffolds to generate human-murine chimeric grafts which are then grafted to full-thickness surgical wounds in immunodeficient mice. The methods described allow for the generation of a human-mouse follicular structure.