Hair follicle (HF) regeneration has received great attention in the last decades due to increased demands for effective cures for many diseases that involve hair loss and skin injuries. The challenges met in current pharmacological and surgical approaches have provoked the efforts in developing more effective therapeutic solutions. Cell-based bioengineering of HFs emerges as a promising strategy to overcome the hurdles in regenerating HFs and achieve a “hairy” feat. The main obstacle is our inability to isolate and propagate human dermal papilla (DP) fibroblasts with inductive capabilities, which are the major mesenchymal cells instructing keratinocytes to build the HF structure. Although DP fibroblasts are known to constitute a dermal niche right at the base of the HF, isolation of human DP fibroblasts from HFs is proven to be technically difficult and inefficient. Isolated DP fibroblasts proliferate slowly under normal culture conditions due to missing key epithelial and microenvironmental cues. Furthermore, human DP fibroblasts quickly lose hair inductivity during ex vivo expansion and fail to produce HFs. The anatomical proximity and shared origin between dermal fibroblasts and DP fibroblasts suggest that dermal fibroblasts may represent a viable option for supplying a large number of inductive DP fibroblasts. However, dermal fibroblasts have not been explored whether they can be reprogrammed to possess DP hair inductivity for HF bioengineering. Our long-term goal is to develop novel bioengineering approaches to produce fully functional human HFs for clinical procedures. Using a three- dimensional (3D) composite spheroid model, we found that the hair inductivity of human DP fibroblasts is greatly improved and leads to the formation of HF-like structure. Furthermore, we identified five major transcription factors (TFs) that are central to DP phenotype and inductivity. Our central hypothesis is that the DP phenotype can be driven in human dermal fibroblasts (DFs) by activating master DP-TFs, thus reprogramming non‐hairy epidermis to a follicular fate. In the first aim, we will determine whether human dermal fibroblasts can be reprogrammed to establish DP characteristics and hair inductivity. We will activate the expression of five major DP inductivity-related TFs in human DFs using a CRISPR activation system. Furthermore, we will identify which TF has the highest transcriptional activity in reprogramming human dermal fibroblasts to initiate DP gene expression, DP-keratinocyte interaction, and DP HF inductivity. In the second aim, we will determine whether reprogrammed dermal fibroblasts in composite spheroids can induce HFs in ESS. We will use a combination of 3D composite spheroid and premade micropores to create an inductive niche in ESS. This work will provide a new and in-depth understanding of novel mechanistic knowledge regarding genetic programs that are critical to promoting DP characteristics and HF morphogenesis. The outcomes are sign...