We have been proposing a label-free cell separation device called "cell rolling column" in which cells selectively roll on antibody-immobilized column surfaces. The aim of this study was to investigate the possibilities of separation of undifferentiated induced pluripotent stem (iPS) cells from heterogeneous cells. Three types of phospholipid polymer, poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-w-butyl methacrylate (BMA)-co-p-nitrophenyl oxycarbonyl poly (ethylene glycol) methacrylate (MEONP)], were synthesized with monomer feed ratios of MPC:BMA:MEOPN of 30:60:10 (PMBN 10), 33:66:1 (PMBN 1) and 33:67:0 (PMBN 0). A microfluidic channel was coated with PMBNs and immobilized with antibodies to stage-specific embryonic antigen 1 (SSEA-1), known as a pluripotency marker for murine cells. Suspended iPS cells were infused into the channels, and cell rolling speed and SSEA-1 expression were investigated. The results indicate that PMBN 10 coated channel showed highest density of anti-SSEA-1 antibody immobilization, and SSEA-1 positive cells were rolled on the surface. It is concluded that the cell rolling column can be a promising tool for a cell separation system for undifferentiated iPS cells.