Lectins are carbohydrate-binding proteins, which occur ubiquitously in nature and are abundant in all living organisms from bacteria to mammals. They have several biological functions among which cell adhesion is well known and characterized. Based on the characterization of the glycome of human embryonic stem cells (hESCs), we have investigated the properties of glycan-binding lectins as a novel class of culture support matrices supporting hESC culture. We report that an Erythrina cristagalli lectin (agglutinin) (ECA) matrix supported the undifferentiated growth and significantly increased the plating efficiency of both hESC and human induced pluripotent stem cells when used in conjunction with pinacidil, an antihypertensive drug with ROCK inhibition activity. As a matrix, ECA maintained pluripotency, robust proliferation with a normal karyotype, and the ability to differentiate both in vitro and in vivo. Therefore, our findings indicate that lectins are potential candidates for design of culture and differentiation methods, and that ECA is a potent simple defined matrix for human pluripotent stem cells.