Hair follicle dermal papilla cells (HFDPC) are a specialized cell population located in the bulge of the hair follicle with unique characteristics such as aggregative behavior and the ability to induce new hair follicle formation. However, when expanded in conventional 2D monolayer culture, their hair inductive potency is rapidly lost. Different 3D culture techniques, including cell spheroid formation, have been described to restore, at least partially, their original phenotype, and therefore, their hair inductive ability once transplanted into a recipient skin. Moreover, hair follicle dermal papilla cells have been shown to differentiate into all mesenchymal lineages, but their differentiation potential has only been tested in 2D cultures. In the present work, we have cultured HFDPC in the 3D self-assembling peptide scaffold RAD16-I to test two different tissue engineering scenarios: restoration of HFDPC original phenotype after cell expansion and osteogenic and adipogenic differentiation. Experimental results showed that the 3D environment provided by RAD16-I allowed the restoration of HFDPC signature markers such as alkaline phosphatase, versican and corin. Moreover, RAD16-I supported, in the presence of chemical inductors, three-dimensional osteogenic and adipogenic differentiation. Altogether, this study suggests a potential 3D culture platform based on RAD16-I suitable for the culture, original phenotype recovery and differentiation of HFDPC.