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Stem cell therapy has emerged as the most vibrant area of research, due to the capacity of stem cells for self-renewal and differentiation into different types of cell lines upon their culture. But lately, scientists become increasingly aware of the limitations of conventional 2D culture and stem cell culture media, due to several key drawbacks associated with this model, such as immune response upon transplantation, animal pathogen contamination, and complication, during developmental studies due to undefined factors in the cultural media. In this study, an attempt has been made to develop a new type of polymeric 3D scaffold based on the self-assembly of a star-like amphiphilic copolymer of poly(caprolactone)–poly(ethylene oxide) unit into nanowires (nanofibers), that have a scale similar to the native extracellular matrix and are capable of mimicking the extracellular microenvironment where the functional properties of stem cells can be observed and manipulated. The obtained data showed that polymeric-based nanofibers can be used as a 3D scaffold for mouse embryonic stem cells (mESCs) growth without losing their stem cell phenotype. The results obtained suggest that the polymeric 3D scaffolds (nanofibers) not only support stem cells’ growth and proliferation but also preserve the mESC pluripotency.