Specification of limb field and initiation of limb development involve multiple steps, each of which is tightly regulated both spatially and temporally. Recent developmental analyses on various vertebrates have provided insights into the molecular mechanisms that specify limb field and have revealed several genetic interactions of signals involved in limb initiation processes. Furthermore, new approaches to the study of the developmental mechanisms of the lateral plate mesoderm of amphioxus and lamprey embryos have given us clues to understand the evolutionary scenarios that led to the acquisition of paired appendages during evolution. This review highlights such recent findings and discusses the mechanisms of limb field specification and limb bud initiation during development and evolution.