Previous reports have indicated that extracellular matrices (ECMs) affect the developmental fate of human embryonic stem cells (hESCs). Specially, type IV collagen and laminin, which belong to a group of macromolecular proteins with a substantial proportion of ECMs, are known to influence the proliferation and differentiation of hES cells. In this study, we evaluated the effects of type IV collagen and laminin in freezing medium on the survival and differentiation rates of hES cells after slow freezing and rapid thawing. The addition of type IV collagen (1 μg/ml) to the freezing medium significantly increased the survival rate of hES cells after thawing compared with that of a control group. The spontaneous differentiation rates of groups treated with type IV collagen (1 μg/ml) or laminin (1 μg/ml) were significantly lower than those of the control group. Frozen-thawed hES cells have currently been cultured for more than 70 passages and retain key properties of hES cells such as morphological characteristics, normal karyotype, marker expression (alkaline phosphatase, SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, Rex-1, and Oct-4), basement membrane–related gene expression, and the potential to differentiate into derivatives of all three germ layers. This new slow freezing method by ECM treatment is a reliable and effective cryopreservation method for pluripotent hES cells.