Objective: Embryonic stem cells (ESCs) are pluripotent cells capable of extensive proliferation while maintaining their potential to differentiate into any cell type. The therapeutic potential of these cells are promising, but in many cases limited by our inability to promote their differentiation. In this study we examined the effect of inducer factors in direct differentiation of mouse ESCs into a neural fate in suspension culture systems. Materials and Methods: Mouse ESCs (Royan B1) were cultivated in suspension to form embryoid bodies (EBs) within 2 days (2d). They were induced using astrocyte-conditioned medium (ACM), retinoic acid (RA, 1μM) and basic fibroblast growth factor (bFGF, 20 ng/ml) for 4 days (2+4d) and were cultured on poly-L-lysine coated dishes up to 5 days (2+4+5d) to differentiate. The expressions of neural specific genes were analyzed by immunocytochemistry and reverse transcription-polymerase chain reaction (RT-PCR). ِِDifferences of means for percentage significance of differentiated EBs were tested by the Mann-Whitney Test and for length and thickness of neural processes by t-test. Results: Retinoic acid increased the percentage of EBs outgrowth with neural morphology and bFGF had synergistic effect (at least p<0.05), while ACM did not influence neural differentiation at all. Immunofluorescence analysis revealed the expression of β-tubulin III in neural cells and that the differentiated neural cells in RA+bFGF group were longer and thicker processes (at least p<0.05). The cells expressed Nestin, Pax6, NF-M, Islet-1, Lim1, and HB9. Conclusion: The results showed that ACM had no effect on neural differentiation from ESCs, however, RA was in favor of this and that bFGF increased neural differentiation synergistically by mechanisms that remain to be defined.