The cellular microenvironment comprises soluble factors, support cells, and components of the extracellular matrix (ECM) that combine to regulate cellular behavior. Pluripotent stem cells utilize interactions between support cells and soluble factors in the microenvironment to assist in the maintenance of self-renewal and the process of differentiation. However, the ECM also plays a significant role in shaping the behavior of human pluripotent stem cells, including embryonic stem cells (hESCs) and induced pluripotent stem cells. Moreover, it has recently been observed that deposited factors in a hESC-conditioned matrix have the potential to contribute to the reprogramming of metastatic melanoma cells. Therefore, the ECM component of the pluripotent stem cell microenvironment necessitates further analysis. In this study we first compared the self-renewal and differentiation properties of hESCs grown on Matrigel™pre-conditioned by hESCs to those on unconditioned Matrigel™. We determined that culture on conditioned Matrigel™ prevents differentiation when supportive growth factors are removed from the culture medium. To investigate and identify factors potentially responsible for this beneficial effect, we performed a defined SILAC MS-based proteomics screen of hESC-conditioned Matrigel™. From this proteomics screen, we identified over 80 extracellular proteins in matrix conditioned by hESCs and induced pluripotent stem cells. These included matrix-associated factors that participate in key stem cell pluripotency regulatory pathways, such as Nodal/Activin and canonical Wnt signaling. This work represents the first investigation of stem-cell-derived matrices from human pluripotent stem cells using a defined SILAC MS-based proteomics approach. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.