Recent advances in the field of embryonic stem cells (ESCs) have fueled great hope for regenerative and cell replacement therapies. Currently, stem cell propagation and directed differentiation are carried out in static cultures (e.g. dishes). Nevertheless, the use of static cultures for the generation of ESCs in quantities satisfying medical needs is impractical. Compared to static cultures, stirred-tank bioreactors are advantageous because they are amenable to scale-up, have simple design, and allow for rigorous control of culture conditions. More important, high cell densities can be achieved in such setups. The use of suspension bioreactors was explored for the expansion of undifferentiated ESCs. Previous reports have suggested that cultivation of undifferentiated ESCs is feasible. Here, self-renewing mouse ESCs were expanded in suspension bioreactors without serum. The presence of serum in the culture medium increases the variability in composition, the overall cost, as well as the chance of cell contamination with animal-derived pathogens. Using alternative media formulations, these cells were able to propagate while maintaining their viability above 80%. To show that the cultured cells were still highly pluripotent after long term exposure to suspension culture, the presence of stem cell markers such as Oct-4, Nanog and SSEA-1 was probed by immunofluorescence imaging and flow cytometry. Using quantitative PCR (qPCR), the RNA levels of the pluripotent genes Oct-3/4 and Rex-1 were found to be comparable between suspension cultures and static controls or suspension controls with serum. In vitro differentiations of these cells were performed to investigate whether cells grown in the absence of serum maintained their ability to give rise to cells of all three germ layers. RNA levels of genes specific to particular cell fates were shown to be upregulated by qPCR compared to uncommitted stem cells. The expression of the corresponding proteins was verified by immunocytochemistry. In addition, spectral karyotyping results confirmed that the cells propagated in long-term suspension cultures did not display an abnormal karyotype. The results support the notion that expansion of uncommitted ESCs in suspension bioreactors and in the absence of serum is possible. Culture systems accommodating the propagation and differentiation of ESCs in a large scale will play an important role in regenerative medicine and stem cell biotechnology.