We developed protocols for isolation and characterization of mesenchymal progenitors from murine dermis. Our protocols are part of a more general isolation procedure starting with neonatal murine skin, which has been described in detail by U. Lichti and coauthors (Nat Protoc 3(5):799-810, 2008). We list Lichti's procedures in an abbreviated form as part of this methods section. Our methods to isolate mesenchymal stem cells are presented as a continuous workflow of isolation and characterization, including flow cytometry, cell survival assays, colony formation assays, immunoblotting, immunostaining, multipotential differentiation assays, and in vivo engraftment. In most cases, the protocols are standard; in others, they were adapted to our particular purpose. We made special emphasis on the use of in vitro three-dimensional cultures to cue mesenchymal progenitors into epidermal cells.