CXCL12 is a pleiotropic chemokine capable of eliciting multiple signal transduction cascades and functions, via interaction with either CXCR4 or CXCR7. Factors that determine CXCL12 receptor preference, intracellular signalling route and biological response are poorly understood but are of central importance in the context of therapeutic intervention of the CXCL12 axis in multiple disease states. We have recently demonstrated that 5T4 oncofoetal glycoprotein facilitates functional CXCR4 expression leading to CXCL12 mediated chemotaxis in mouse embryonic cells. Using wild type (WT) and 5T4 knockout (5T4KO) murine embryonic fibroblasts (MEFs), we now show that CXCL12 binding to CXCR4 activates both the ERK and AKT pathways within minutes, but while these pathways are intact, they are non-functional in 5T4KO cells treated with CXCL12. Importantly, in the absence of 5T4 expression, CXCR7 is upregulated and becomes the predominant receptor for CXCL12, activating a distinct signal transduction pathway with slower kinetics involving transactivation of the epidermal growth factor receptor (EGFR), eliciting proliferation rather than chemotaxis. Thus the surface expression of 5T4 marks the use of the CXCR4 rather than the CXCR7 receptor, with distinct consequences for CXCL12 exposure, relevant to the spread and growth of a tumour. Consistent with this hypothesis, we have identified human small cell lung carcinoma cells with similar 5T4/CXCR7 reciprocity that is predictive of biological response to CXCL12 and determined that 5T4 expression is required for functional chemotaxis in these cells.