Amplification of MYCN in neuroblastoma strongly correlates to unfavorable outcome, but little is known of how the high MYCN expression translates into an aggressive tumor phenotype. More aggressive neuroblastomas are generally immature and overexpression of exogenous MYCN in cultured neuroblastoma cells and other neuronal cell types has been reported to inhibit induced differentiation, suggesting a link between high MYCN expression and an immature phenotype. However, we show here that MYCN is expressed in human neuroblasts of sympathetic chain ganglia at fetal week 8.5, a developmental stage at which these neuroblasts express a number of sympathetic neuronal differentiation marker genes. Analyses of 28 neuroblastoma tumor specimens and 27 cell lines for the expression of MYCN and a panel of neuronal differentiation marker genes did not reveal any correlation between MYCN and marker gene expression levels. Finally, we tested five separate differentiation protocols and show that MYCN overexpressing neuroblastoma cells with a neuronal phenotype, derived from the non-MYCN-amplified human neuroblastoma cell line SK-N-SH, retain their capacity to differentiate despite constitutive MYCN overexpression. Our results show that high MYCN expression and sympathetic differentiation are compatible, and indirectly our findings lend support to previously published MYCN neuroblastoma tumor data, which suggest that in single MYCN copy neuroblastomas there is no direct correlation between a high cellular MYCN protein content and aggressive tumor cell behavior.