Limb skeletal elements originate from the limb progenitor cells, which undergo expansion and patterning to develop each skeletal element. Posterior-distal skeletal elements, such as the ulna/fibula and posterior digits develop in a Sonic hedgehog (Shh)-dependent manner. However, it is poorly understood how anterior-proximal elements, such as the humerus/femur, the radius/tibia and the anterior digits, are developed. Here we show that the zinc finger factors Sall4 and Gli3 cooperate for proper development of the anterior-proximal skeletal elements and also function upstream of Shh-dependent posterior skeletal element development. Conditional inactivation of Sall4 in the mesoderm before limb outgrowth caused severe defects in the anterior-proximal skeletal elements in the hindlimb. We found that Gli3 expression is reduced in Sall4 mutant hindlimbs, but not in forelimbs. This reduction caused posteriorization of nascent hindlimb buds, which is correlated with a loss of anterior digits. In proximal development, Sall4 integrates Gli3 and the Plzf-Hox system, in addition to proliferative expansion of cells in the mesenchymal core of nascent hindlimb buds. Whereas forelimbs developed normally in Sall4 mutants, further genetic analysis identified that the Sall4-Gli3 system is a common regulator of the early limb progenitor cells in both forelimbs and hindlimbs. The Sall4-Gli3 system also functions upstream of the Shh-expressing ZPA and the Fgf8-expressing AER in fore-and hindlimbs. Therefore, our study identified a critical role of the Sall4-Gli3 system at the early steps of limb development for proper development of the appendicular skeletal elements.Sall4 | Gli3 | limb progenitors | appendicular skeletal elements | Plzf-Hox H ow progenitor cells are spatially and temporarily organized to construct an organ is a central question in developmental biology. Limb skeletal elements develop from limb progenitors, which arise from the lateral plate mesoderm (LPM) that is originated from epithelial somatopleure (1). Limb progenitors initially form two paired protrusions, fore-and hindlimb buds, whose initiation occurs around embryonic day (E) 9.0 and E9.5, respectively, in mouse embryos. In the following steps, limb signaling centers, known as the zone of polarizing activity (ZPA) and apical ectodermal ridge (AER), are established. SHH (Sonic hedgehog) from the ZPA and FGF8 from the AER are major signal molecules that regulate proliferative expansion and patterning of early limb progenitor cells (reviewed in ref. 2). These processes lead to development of functional limbs with each skeletal element adopting a unique shape at a distinct location.Several studies suggest that limb progenitors consist of two distinct pools, an anterior progenitor pool and a posterior progenitor pool. The posterior progenitor pool consists of cells that once expressed Shh and cells that received paracrine effects of SHH, which contribute to digit 2 (d2), d3, d4, and d5 and the posterior zeugopod (ulna, fibula) (3-6). Contrary to this, the anterior ...