In-vitro investigation of neuronal development and function was initially performed via the isolation and short-term culture of primary neuronal cells. Since then, immortalised neuronal cell lines have allowed for more efficient and reproducible in-vitro neuronal models, bypassing the limited life span of primary neuronal cells. These cell lines can be rapidly differentiated into neuronal cells by altering the cell-culture environment, but do not reach the maturity or diversity of neurons. More recently, the induction of pluripotent stem cells (PSCs) has allowed for in-vitro differentiation of several types of mature neurons, either by directed differentiation media or more efficiently through recombinant expression of transcription factors (TFs). Whilst PSCs offer advantages for mature neuronal models, immortalised neuronal cell lines are widely used and are much simpler to culture. Here we investigated an intermediary option, testing if the same TFs used to induce neurons using PSCs would enhance the differentiation of immortalised neuronal cells. We found that induced differentiation by TF Ngn2 using the piggyBac transposon system improved the differentiation potential of NG108-15 and SH-SY5Y cells towards a cholinergic phenotype. The co-culture of C2C12-derived myotubes showed improved neuromuscular junction formation and myotube differentiation. Alternatively, Ngn2 insertion at the AAVS1 site in SH-SY5Y cells resulted in a mixed cholinergic and glutamatergic phenotype. Furthermore, induced differentiation of NG108-15 cells by TFs Ascl1/Dlx2 resulted in a mixed cholinergic and GABAergic phenotype. Therefore, the use of transcription factors to differentiate immortalised cell lines offers a convenient and efficient alternative for in-vitro models of the nervous system.