Materials for three-dimensional cultures aim to reproduce the function of the extracellular matrix, enabling cell adhesion and growth by remodeling the environment. However, embryonic stem cells (ESCs) must develop in environments that prevent adhesion and preserve their pluripotency. In this study, we used cellulose nanofiber hydrogels to mimic the developing conditions required for ESCs. These plant-based hydrogels are simultaneously biocompatible and exogenous to mammalian cells, preventing remodeling and attachment. The storage modulus of these hydrogels could be fine-tuned by varying the degree of oxidation to enable selective degradation. The ESCs proliferated in the artificial environment, forming increasingly large embryoid bodies for 15 days. Unlike traditional cultures in which ESCs begin differentiating upon the removal of the chemical inhibition, the expression of pluripotency markers in the ESC population remained high for the entire two weeks. Cellulase from Trichoderma reesei was used to retrieve the ESC cultures selectively. The proposed unique system is a prospective model with which to study the early development of embryonic cells, as well as a nonchemical method of preserving undifferentiated populations of ESCs.