Fibroblasts, which are a major component of cancer-induced stroma, can have a significant impact on the progression of adjacent malignant epithelia. To characterize fibroblasts recruited into cancer-induced stroma, we examined the recruitment efficiency of 9 human fibroblast cell lines into experimental tumors generated in immunodeficient mice. Green fluorescence protein (GFP)-labeled fibroblast cell lines and human pancreatic cancer cell line Capan-1 were injected i.p. at different sites; the GFP-labeled cells within xenografts were then analyzed. KM104GFP (bone marrow) and VA-13GFP (lung) were selectively recruited into cancer stroma more efficiently than the other cell lines. KM104GFP cells did not affect tumor volume; however, VA-13GFP cells increased tumor volume by about 2-fold. After 5 cyclic in vivo passages of KM104GFP in Capan-1, we selected a subpopulation with an 8.4-fold higher recruitment efficiency (KM104GFP-5G) compared to parental KM104GFP. KM104GFP-5G also exhibited higher chemotaxis and chemoinvasion activity compared to KM104GFP in response to cancer-released chemoattractant(s). Oligonucleotide microarray analysis identified 8 genes with >3-fold upregulation and 6 genes with >3-fold downregulation in KM104GFP-5G. Immunohistochemistry confirmed that fibroblasts recruited into pancreatic cancer stroma strongly expressed carbonic anhydrase IX and keratin-8, whose transcripts were upregulated in KM104GFP-5G by oligonucleotide microarray analysis, whereas their expression in fibroblasts within noncancerous pancreatic stroma were under the detection level. Our results indicate that fibroblast recruitment is not selective with respect to organ origin and that particular fibroblast subpopulations with specific phenotypic characteristics could be recruited efficiently into cancer-induced stroma.