The murine embryonic stem cell (ESC) in vitro differentiation system was used to study the role of interleukin-6 (IL-6) in the induction of early embryonic hematopoietic development. When cultured in human umbilical cord serum (HUCS), pluripotent ESCs differentiate to embryoid bodies expressing tissue development equivalent to day 7-10 mouse embryos. Pooled HUCS samples were found to contain 10 to 13 pg/mL IL-6. Depletion of IL-6 from HUCS decreased the percentage of differentiated embryoid bodies containing hematopoietic islands, and decreased the number of hematopoietic foci per embryoid body as well. Although pooled samples of HUCS also contained granulocyte colony-stimulating factor (G-CSF), depletion of G-CSF from HUCS had no effect on either the percentage of embryoid bodies containing hematopoietic foci or the number of hematopoietic foci per embryoid body. These data suggest that IL-6 is one important cytokine in the inductive differentiation of pluripotent ESCs toward hematopoiesis in vitro.