The ability of embryonic stem cells (ESCs) to differentiate into all somatic cell types makes them an attractive cell source for regenerative medicine and tissue-engineering applications. In addition to their potential to restore cellularity of injured or diseased tissues, molecular factors produced by stem cells may also directly influence tissue morphogenesis, thereby providing therapeutic benefit independent of stem cell differentiation. In order to examine this hypothesis, it is necessary to separate the cells from the molecular factors they are capable of producing. One potential method of separation is to acellularize clusters of differentiating ESCs, referred to as embryoid bodies (EBs), from the extracellular matrix they synthesize. Thus, the objective of this study was to examine the effectiveness of different reagents, including peracetic acid, sodium dodecyl sulfate, Triton X-100 and DNase, to acellularize EBs. The efficiency of acellularization was assessed based on cell viability and retention of overall mass, DNA and protein, as well as histological examination of the resulting acellular matrix. Initial studies suggested that sequential treatments of Triton X-100 and DNase successfully yielded a cohesive acellular product that retained protein content and significantly reduced levels of DNA. Additional optimization studies were performed with combinations of Triton X-100 and DNase to assess the specific effects of reagent concentration, treatment duration and solvent volume/EB ratios. These results establish methods to effectively obtain novel acellular matrices from differentiating ESCs that may contain morphogenic cues and have potential applications in regenerative medicine.