Erythropoietin (Epo) is known to have a significant role in tissues outside the hematopoietic system. In this work, we investigated the function of Epo in cells of neuronal origin subjected to differentiation. Treatment of SH-SY5Y cells with all-trans-retinoic acid (atRA) generated differentiated neuron-like cells, observed by increased expression of neuronal markers and morphological changes. Exposure of undifferentiated cells to proapoptotic stimuli such as staurosporine, TNF-α, or hypoxia, significantly increased programmed cell death, which was prevented by previous treatment with Epo. In contrast, atRA-differentiated cultures showed cell resistance to apoptosis. No additional effect of Epo was detected in previously differentiated cells. The inhibition of the PI3K/Akt pathway by Ly294002 abrogated the protective effects induced by either Epo or atRA. The effect of atRA was mediated by an increased expression of Bcl-2 whereas the Epo treatment upregulated not only Bcl-2 but also Bcl-xL. This upregulation by Epo was not detected in atRA-differentiated cells, thus confirming the lack of the protective effect of Epo. As expected, assays with AG490, an inhibitor of Jak2, blocked the Epo action only in undifferentiated cells. This reduced neuroprotective function of Epo on SH-SY5Y differentiated cells could be explained at least in part by downregulation of the Epo receptor expression, which was observed in atRA-differentiated cells. This study shows differential cellular protection induced by Epo at two stages of SH-SY5Y differentiation. The results allow us to suggest that this differential cell behavior can be ascribed to the interaction between atRA and the signaling pathways mediated by Epo. J. Cell. Biochem. 110: 151–161, 2010. © 2010 Wiley-Liss, Inc.