Little is known about the growth-signaling pathways that govern the proliferation of Ewing tumor (ET) cells either in vitro or in vivo. We have studied signal transduction pathways in ET cell lines and compared kinase expression levels and proliferation rates with primary tumors. Cell lines were studied both as conventional adherent monolayers and as anchorage-independent multi-cellular spheroids. Importantly, we observed significant differences between these in vitro models and found that ET spheroids were more closely related to primary tumors with respect to cell morphology, cell-cell junctions, proliferative index and kinase activation. Monolayer ET cells demonstrated serum-dependent phosphorylation of ERK1/2 and AKT and constitutively high serum-independent cyclin D1 protein expression. However, when ET cells were placed in suspension culture, there was immediate serum-independent activation of ERK1/2 and AKT. In addition, cyclin D1 protein expression was completely blocked until stable multi-cellular spheroids had formed, indicating that cell-cell adhesion is necessary for the proliferation of anchorage independent ET cells. This reduction in cyclin D1 expression was post-transcriptional and could be mimicked in monolayer cells by treatment with phosphatidyl inositol-3 kinase (PI3K) inhibitors. Moreover, PI3K inhibition significantly reduced ET cell proliferation and, in primary ET samples, cyclin D1 expression correlated with expression of activated AKT. Thus, the PI3K-AKT pathway appears to be critical for the proliferation of ET cells both in vitro and in vivo and tumor cell growth in vivo may be better represented by the study of anchorage-independent multi-cellular spheroids.