Experimental manipulation of hESCs has been hampered by their fragility and susceptibility to apoptosis when dissociated into single cells. The OxF lines are particularly robust and may be successfully passaged as single cells, with the inclusion of ROCK inhibitor in the medium. The protocols here describe the enzymatic dissociation of hESCs into a single-cell suspension and the plating of these cells onto either feeder cells or a protein-coated surface.