Homeobox-containing genes play an important role in patterning processes that occur during embryogenesis. Programmed cell death is a key process during pattern formation. The mechanisms by which programmed cell death is spatially regulated are not well characterized. Msx1 and Msx2 are two closely related homeobox-containing genes that are expressed at sites where cellular proliferation and programmed cell death occur, including the developing limb and the cephalic neural crest. Tissue interactions are necessary for the maintenance of Msx1 and Msx2 expression and programmed cell death. It has been demonstrated that BMP4 can regulate cell death at these same sites as well as induce Msx expression. These observations lead to the hypothesis that Msx2 is a key regulator of cell death in the BMP-mediated pathway. Embryonic stem cell lines will undergo processes typical of early embryogenesis upon aggregation and have recently been shown to provide a model system for programmed cell death. In contrast to ES cells, we see that P19 cells do not undergo pronounced cell death upon aggregation; however, constitutive ectopic Msx2 expression in P19 cells results in a marked increase in apoptosis induced upon aggregation but has no effect when cells are grown as a monolayer. If aggregates are allowed to interact with a substrate, the process of programmed cell death is completely inhibited. Addition of BMP4 to aggregated P19 cells also results in cell death; however, BMP4 does not increase levels of cell death in Msx2-expressing cells. Addition of BMP4 to P19 cells results in an induction of Msx2 transcription consistent with its proposed role in cell death in the embryo. Our data support a model by which BMP4 induces programmed cell death via an Msx2-mediated pathway and provide direct functional evidence that Msx2 expression is a regulator of this process.