The spleen is a vertebrate organ that has both hematopoietic and immunologic function. The embryonic origins of the spleen are obscure, with most studies describing the earliest rudiment of the spleen as a condensation of mesodermal mesenchyme on the left side of the dorsal mesogastrium. The development of spleen handedness has not been described previously, presumably because of the difficulty in assaying spleen position in the embryo and the lack of early, organ-specific molecular markers. Here we show that expression of the homeobox gene Nkx2-5 serves as a marker for spleen precursor tissue. Pre-splenic tissue is initially located in symmetric domains on both sides of the embryo but, during subsequent development, only the left side goes on to form the mature spleen. Therefore, the final location of the spleen on the left side of the body axis appears to result from preferential development of the spleen precursor cells on the left side of the embryo. Our studies indicate that the spleen and heart become asymmetric via different cellular mechanisms. Nkx2-5 may function locally as part of the laterality cascade, downstream of nodal and Pitx2, or it may direct asymmetric morphogenesis after laterality has been determined.