Embryonic stem cells (ESCs) are pluripotent cells derived from the inner cell mass of the blastocyst that can give rise to cells of the ectoderm, endoderm and mesoderm lineages. Once isolated from the blastocyst, ESCs can be cultured indefinitely in vitro in an undifferentiated state or can be induced to differentiate. In the case of mouse ESCs (mESCs), the cytokine leukemia inhibitory factor (LIF) is added to culture media to maintain pluripotency and is removed to induce differentiation. Although it is known that extracellular matrix (ECM) components influence stem cell maintenance, proliferation and differentiation, the precise effects of ECM environments on embryonic stem cell behavior have not been systematically studied. The main purpose of this thesis project was to investigate the behavior of undifferentiated mESCs cultured in different 3D hydrogel matrices and to determine whether viscoelastic and biochemical variations in the matrices differentially affect the ability of stem cells to self-renew; that is, retain their pluripotency or undifferentiated phenotype. Their behavior in 3D environments was compared to mESC behavior in traditional 2D culture. In addition, a new method of casting hydrogels in polydimethylsiloxane (PDMS) molds was developed in order to efficiently cast multiple hydrogels of varying sizes and shapes. The findings of this thesis project will benefit both the scientific and engineering community as it encourages researchers to re-evaluate the quality of standard 2D embryonic stem cell culture methods versus potentially novel and advantageous 3D hydrogel culture methods.