AbstractAmong the different parameters governing the successful derivation and expansion of human embryonic stem cells (hESC), feeder layers play the most important role. Human feeders in form of human mesenchymal stromal cells (hMSCs) and human foreskin fibroblasts (HFFs) lay the foundation for eradication of animal‐derived hESC culture system. In this study we explored the potential of human foreskin derived mesenchymal like stromal cells (HF‐MSCs) to support self renewal and pluripotency of hESC. The MSCs isolated from human foreskin were found to be resistant to standard concentrations and duration of mitomycin‐C treatment. Growth pattern, gene profiling (Oct‐4, Nanog, Sox‐2, Rex‐1), cytoskeletal protein expression (vimentin, nestin) and tri‐lineage differentiation potential into adipocytes, chondrocytes and osteocytes confirmed their mesenchymal stromal cell status. Further, the HF‐MSCs were positive for CD105, CD166, CD73, CD44, CD90, SSEA‐4, and negative for CD34, CD45, HLA‐DR cell‐surface markers and were found to exhibit BM‐MSC‐like characteristics. hESC lines co‐cultured with HF‐MSC feeders showed expression of expected pluripotent transcription factors Oct‐4, Nanog, Sox‐2, GDF‐3, Rex‐1, STELLAR, ABCG2, Dppa5, hTERT; surface markers SSEA‐4, TRA‐1‐81 and maintained their cytogenetic stability during long term passaging. These novel feeders also improved the formation of embryoid bodies (EBs) from hESC which produced cell types representing three germ layers. This culture system has the potential to aid the development of clinical‐grade hESCs for regenerative medicine and drug screening. Further, we envisage foreskin can serve as a valuable source of alternative MSCs for specific therapeutic applications. J. Cell. Biochem. 112: 1353–1363, 2011. © 2011 Wiley‐Liss, Inc.