The skin is the outermost barrier organ in the body, which contains several types of dendritic cells (DCs), a group of professional antigen-presenting cells. When the skin encounters invading pathogens, different cutaneous DCs initiate a distinct T cell immune response to protect the body. Among the invading pathogens, fungal infection specifically drives a protective interleukin-17-producing Th17 immune response. A protocol was developed to efficiently differentiate Th17 cells by intradermal Candida albicans infection to investigate a subset of cutaneous DCs responsible for inducing Th17 immunity. Flow cytometry and gene expression analyses revealed a prominent induction of Th17 immune response in skin-draining lymph nodes and infected skin. Using diphtheria toxin-induced DC subset-depleting mouse strains, CD301b+ dermal DCs were found to be responsible for mounting optimal Th17 differentiation in this model. Thus, this protocol provides a valuable method to study in vivo function of differential subsets of cutaneous DCs to determine Th17 immunity against deep skin fungal infection.