Sef and Sprouty proteins function as feedback antagonists of fibroblast growth factor (Fgf) signaling in zebrafish embryos. To study the role of Sef in mice, we generated Sef homozygous mutant animals. These animals are viable and show normal expression of mid-hindbrain genes at embryonic days 8.5 and 9.5. To investigate the possibility of functional synergism between Sef and Sprouty proteins, we electroporated Sprouty2Y55A, which functions in a dominant-negative manner in tissue culture cells into the mid-hindbrain region of wildtype and Sef mutant embryos. The expression pattern of Gbx2, a downstream target of Fgf signaling, was expanded or shifted in electroporated embryos, and this effect was significantly enhanced in the Sef mutant background. Altogether, our results demonstrate that Sef and Sproutys function synergistically to regulate Gbx2 expression in the anterior hindbrain. genesis 41:110–115, 2005. © 2005 Wiley-Liss, Inc.