We report a novel method for culturing human embryonic stem cells (HES) using 300-μm-thick acellular kidney-derived microscaffolds (KMSs) that allow cells to obtain nutrients and gasses simply by diffusion, enabling the KMSs to be used readily ex vivo under defined culture conditions, without the need for vascularization/perfusion or transplantation. Standard histology and scanning electron microscopy show that HES grow and expand according to the complex structure dictated by the scaffold. We further show that the expression levels of NPHS-1, REN, AQP-1, SLC2A2, and ANPEP were 7.6-, 5.1-, 128-, 4.3-, and 3.9-fold higher, respectively, when the HES were grown on KMS compared with the HES grown on collagen. Similarly, the levels of these genes were 10-, 30-, 4.6-, 7.5-, and 3-fold higher, respectively, when the HES were grown on KMS compared with the HES grown on Matrigel. We have also shown that culturing HES in 5% kidney-derived serum-free conditioned media can lead to the upregulation of NPHS-1, REN, and EPO by 3-, 18-, and 15-fold, respectively. This article demonstrates a novel way of growing HES in vitro whereby the beneficial biophysical as well as biochemical surroundings of the seeded cells provide a more in vivo-like environment, thereby assisting in differentiation of the HES toward a renal lineage. The approach presented here may provide a powerful tool for in vitro study of HES differentiation toward kidney-specific cell lineages under controlled conditions.