Neonatal gonocytes are direct precursors of spermatogonial stem cells, the cell pool that supports spermatogenesis. Although unipotent in vivo, gonocytes express pluripotency genes common with embryonic stem cells. Previously, we found that all-trans retinoic acid (RA) induced the expression of differentiation markers and a truncated form of platelet-derived growth factor receptor (PDGFR)β in rat gonocytes, as well as in F9 mouse embryonal carcinoma cells, an embryonic stem cell-surrogate that expresses somatic lineage markers in response to RA. The present study is focused on identifying the signaling pathways involved in RA-induced gonocyte and F9 cell differentiation. Mitogen-activated protein kinase kinase (MEK) 1/2 activation was required during F9 cell differentiation towards somatic lineage, whereas its inhibition potentiated RA-induced Stra8 expression, suggesting that MEK1/2 acts as a lineage specification switch in F9 cells. In both cell types, RA increased the expression of the spermatogonial/premeiotic marker Stra8, which is in line with F9 cells being at a stage before somatic-germline lineage specification. Inhibiting PDGFR kinase activity reduced RA-induced Stra8 expression. Interestingly, RA increased the expression of PDGFRα variant forms in both cell types. Together, these results suggest a potential cross talk between RA and PDGFR signaling pathways in cell differentiation. RA receptor-α inhibition partially reduced RA effects on Stra8 in gonocytes, indicating that RA acts in part via RA receptor-α. RA-induced gonocyte differentiation was significantly reduced by inhibiting SRC (v-src avian sarcoma [Schmidt-Ruppin A-2] viral oncogene) and JAK2/STAT5 (Janus kinase 2/signal transducer and activator of transcription 5) activities, implying that these signaling molecules play a role in gonocyte differentiation. These results suggest that gonocyte and F9 cell differentiation is regulated via cross talk between RA and PDGFRs using different downstream pathways.