To understand factors that regulate leukocyte entry and positioning within human melanoma tissues, we performed a multi-parametric quantitative analysis of two separated regions: the intratumoral area and the peritumoral stroma. Using two mesenchymal markers, Fibroblast Activation Protein (FAP) and CD90, we identified three subsets of mesenchymal cells (MC): (i) intratumoral FAP+CD90low/- MC, (ii) peritumoral FAP+CD90+ MC, and (iii) FAP-CD90+ perivascular MC. We characterized CD90+ MC, which showed a stable CCL2 secretory phenotype when long-term expanded ex vivo, and heavily surrounded peritumoral DARC+ postcapillary venules, supporting a role for these vessels in peritumoral inflammatory leukocyte recruitment. Conversely, the intratumoral area was variably invaded by FAP+CD90low/- MC that colocalized with a distinct extracellular matrix network. A positive correlation was observed between intratumoral stromal cell/ECM networks and leukocyte infiltration among tumor-cells, as well as in a stroma-dependent xenograft tumor model. Adoptively transferred T lymphocytes preferentially infiltrated tumors composed of TC+MC, compared to TC only. Altogether our results suggest that a variety of MC contribute to regulate different steps of leukocyte tumor infiltration; i.e. CD90+ cells surrounding peritumoral vessels secrete CCL2 to recruit CCR2+ leukocytes at the tumor periphery, whereas intratumoral FAP+ cells organize a stromal-scaffold that contact-guide further invasion among dense-packed tumor cells.Journal of Investigative Dermatology accepted article preview online, 27 February 2013; doi:10.1038/jid.2013.88.