We have investigated the structural basis by which the counterclockwise direction of the amniote gut is established. The chirality of midgut looping is determined by left-right asymmetries in the cellular architecture of the dorsal mesentery, the structure that connects the primitive gut tube to the body wall. The mesenchymal cells of the dorsal mesentery are more condensed on the left side than on the right and, additionally, the overlying epithelium on the left side exhibits a columnar morphology, in contrast to a cuboidal morphology on the right. These properties are instructed by a set of transcription factors: Pitx2 and Isl1 specifically expressed on the left side, and Tbx18 expressed on the right, regulated downstream of the secreted protein Nodal which is present exclusively on the left side. The resultant differences in cellular organization cause the mesentery to assume a trapezoidal shape, tilting the primitive gut tube leftward.