Human embryonic stem (ES) cell culture has developed over the years allowing the subtle procedures that are easy to manipulate. Feeder-free ES cell culture is an important milestone for human pluripotent stem cell research which eliminates the feeder cells. Various matrices and medium formulations have been generated for feeder-independent culture. Here we described an mTeSR™1 based feeder-independent human ES cell culture on Matrigel® matrix. Culture, freeze/thaw, passaging and initiation of differentiation in suspension culture were described.