Langerhans cells (LCs) are highly abundant dendritic cells (DCs) in epidermal and mucosal tissues. The transcription factors PU.1 and Id2 have been implicated as positive regulators of LC development from hematopoietic progenitor cells. LC differentiation from progenitors is absolutely dependent on transforming growth factor beta 1 (TGF-beta1) in vitro as well as in vivo; however, downstream mechanisms are poorly defined. We found that both PU.1 and Id2 are induced by TGF-beta1 in human CD34+ monocyte/LC (M/LC) progenitor cells, and that neither ectopic PU.1 or Id2 alone, nor both together, could replace TGF-beta1 in its instructive function on LC commitment. However, both factors critically contributed to LC differentiation by acting at 2 distinct intersection points. Ectopic PU.1 strongly enhanced TGF-beta1-dependent LC development. Additionally, Notch-induced generation of interstitial-type DCs was associated with PU.1 up-regulation. Thus, PU.1 is generally increased during myeloid DC development. Ectopic Id2 inhibits the acquisition of early monocytic characteristics by cells generated in the absence of TGF-beta1 and also inhibits monocyte induction by alternative stimuli. Since TGF-beta1 represses a default monocyte pathway of common progenitor cells, PU.1 and Id2 seem to modulate lineage options of M/LC precursors, downstream of TGF-beta1.