In the developing vertebrate brain, the boundary separating the midbrain from the hindbrain forms through a highly conserved folding of the neuroepithelial tissue, known as the midbrain-hindbrain boundary (MHB) constriction. During MHB formation, cells at the constriction shorten and narrow before basally constricting and apically expanding to form a sharp fold in the tissue. We previously determined that two isoforms of the actin cytoskeletal motor protein, non-muscle myosin II (NMII), differentially regulate cell shape, with non-muscle myosin IIA specifically regulating cell length and non-muscle myosin IIB specifically regulating cell width. Non-muscle myosin II proteins are tightly regulated via the phosphorylation of their associated myosin regulatory light chains; however, the upstream signaling pathways that initiate differential regulation of NMII-mediated cell shape during midbrain-hindbrain boundary morphogenesis are not known. Our current studies have revealed that calcium signaling is critical for the regulation of cell length, but not cell width, during midbrain-hindbrain boundary formation. In particular, manipulation of cytosolic calcium levels resulted in abnormal midbrain-hindbrain boundary cell length and inhibition of cytosolic calcium rescued the cell length phenotype observed in embryos with over activation of non-muscle myosin II. In addition, we found that calcium signals mediate phosphorylation of myosin light chain in the midbrain-hindbrain boundary region. In addition, we have found that wnt5b, which is expressed specifically at the MHB during the time of morphogenesis, is an upstream regulator of cell width. 2D Differential Gel Electrophoresis also revealed that Wnt5b potentially regulates tubulin levels, implicating a novel role for microtubules in this process. Together our findings suggest that modulation of myosin activity by calcium and Wnt5b are critical for proper regulation of cell length and cell width at the MHB to determine embryonic brain shape. We further hypothesize that these mechanisms may be conserved and critical for shaping other epithelial cells and tissues throughout development.