Neural transcription factors are important for the differentiation and integration of neurons. Reprogramming neural cells to increase expression of the transcription factor NeuroD1 is a potential therapeutic approach to treat neurodegenerative diseases through increased local neurogenesis. Using direct reprogramming to transdifferentiate astrocytes into neurons results in downregulation of astrocyte specific promoters such as GFAP and subsequent loss of vector-related fluorescent markers. Dual AAV Cre-FLEX vectors lock their target genes in an ‘activated’ orientation, providing clearer evidence on whether reported effects on neurogenesis result from AAV reprogramming or spared neuronal populations. Using a serotype-five AAV (AAV-5) Cre^GFAP-FLEX ^NeuroD1-GFP system that specifically links to the GFAP promoter of reactive astrocytes, NeuroD1 reportedly directs astrocyte transdifferentiation towards neuronal phenotypes. We used supposedly pure fetal human astrocytes or neural progenitor cell (NPC) cultures to determine the specificity of the Cre^GFAP-FLEX ^NeuroD1-GFP AAV-5 system for astrocytes, and whether it causes transdifferentiation towards a neuronal phenotype. After one and six days there was no obvious GFP-fluorescence or consistent change in cellular morphology in either ‘astrocyte’ or NPC cultures. Instead, after plating commercially available fetal cell lines, a surprising heterogenous cell culture was identified by fluorescent protein markers in control and experimental trials, indicating limitations of the Cre-FLEX AAV-5 system. Namely, that the CRE-FLEX system requires both the Cre and FLEX AAVs to transfect individual cells to be effective, that the AAV-5 serotype may not be suitable for transfecting astrocytes, and that the presence of heterogeneous cell cultures may generate misleading result interpretation.