There is only little information about sulfated polysaccharides relevance as a factor of microenvironment for stem cell differentiation. The aim of present study was to test the impact of dextran sulfate and levan sulfate, a synthetic derivative of bacterial polyfructan levan, on embryonic stem cell differentiation in vitro using mouse CGR8 embryonic bodies (EBs) as model system. The differentiation of cardiomyocytes, erythroid cells, leukocytes and endothelial cells was evaluated by light microscopy and RT-PCR. In the presence of levan and dextran sulfates an increase of the number of EBs with hemoglobin-containing erythroid cells was observed, and enhanced bH1-globin and bmajor-globin mRNA expression was demonstrated. Maximum effects were achieved at polysaccharide concentrations ranging from 100 to 500 mg/ml at 7–10 days after plating of 6-day-old EBs on cell culture dishes. RT-PCR with PECAM-1 and CD-45 primers evidenced stimulation of endothelial cell and leukocytes differentiation upon treatment with sulfated levan and sulfated dextran. Sulfated polysaccharides at high concentrations (500 mg/ml) exerted inhibiting effects on cardiomyocyte differentiation. In summary, our data indicate that sulfated polysaccharides stimulated differentiation of the hematopoietic/endothelial cell lineage and depressed cardiomyogenesis of ES cells.