Human male germ cell tumors (GCTs) comprise an excellent model system for understanding the molecular events controlling cellular differentiation and lineage decision. Pluripotential embryonal carcinoma cell lines derived from GCTs can be induced to undergo terminal differentiation along specific lineages dependent upon the differentiating agent. We report here that one such cell line, NTera2/clone D1 (NT2/D1), previously shown to undergo differentiation along a neuronal lineage by all-trans-retinoic acid (RA), can be induced along a distinct non-neuronal lineage by the mammalian morphogens, bone morphogenetic proteins-2 and -4 (BMP-2 and -4). Very little is known regarding the molecular events that govern such human lineage decisions. In this study, the role of the ID (inhibitor of differentiation and DNA-binding) family of genes that act as inhibitors of the function of helix-loop-helix (HLH) transcriptional activators involved in lineage commitment was investigated using two pluripotential GCT cell lines as a model system. In the differentiation programs studied, Id1 was noted to decline, an event often associated with the decrease in proliferative rate occurring during differentiation. However, differences in the expression of ID2 and ID3 family members were detected between the programs. Notably, an increase in Id3 during RA-induced differentiation of NT2/D1 cells was observed, while Id2 levels increased during BMP-2 and -4 treatment of NT2/D1 cells and during the induction of an endodermal-like differentiation program in the cell line, 27X-1. The pluripotential male GCT cell lines comprise a unique system in which the roles of specific genes such as the ID family of genes in human cell differentiation and lineage decision can be studied.