The dermal papilla (DP) plays pivotal roles in hair follicle morphogenesis and cycling. However, characterization and/or propagation of human DP have been unsatisfactory because of the lack of efficient isolation methods and the loss of innate characteristics in vitro. We hypothesized that culture conditions sustaining the intrinsic molecular signature of human DP could facilitate expansion of functional DP cells. To test this, we first characterized the global gene expression profile of microdissected, non-cultured human DPs. We performed a “two-step” microarray analysis to exclude the influence of unwanted contaminants in isolated DPs and successfully identified 118 human DP signature genes, including 38 genes listed in the mouse DP signature. The bioinformatics analysis of the DP gene list revealed that WNT, BMP and FGF signaling pathways were upregulated in intact DP and addition of 6-bromoindirubin-3′-oxime, recombinant BMP2 and basic FGF to stimulate these respective signaling pathways resulted in maintained expression of in situ DP signature genes in primarily cultured human DP cells. More importantly, the exposure to these stimulants restored normally reduced DP biomarker expression in conventionally cultured DP cells. Cell growth was moderate in the newly developed culture condition. However, rapid DP cell expansion by conventional culture followed by the restoration by defined activators provided a sufficient number of DP cells, which demonstrated characteristic DP activities in functional assays. The present study revealed previously unreported molecular mechanisms contributing to human DP properties and describes a useful technique for the investigation of human DP biology and hair follicle bioengineering.