Human neural stem cells (hNSCs) have long been used as an in vitro model to study neurogenesis and as candidates for nervous system therapy. Many parameters have been considered when evaluating the success of transplantation, but sex of donor and recipients is often not discussed. We investigated two commercial NSC lines, the female hNSC-H9 and male hNSC-H14, and we observed faster growth rates in the male cells. At 4 days of differentiation, male cells presented a significant increase in expression of DCX, an immature neuronal marker, while female cells showed a significant increase in RMST, a long noncoding RNA, which is indispensable during neurogenesis. In addition, expression of neural markers MAP2, PSD95, SYP, DCX, and TUJ1 at day 14 of differentiation suggested a similar differentiation potential in both lines. The most significant differences at day 14 of differentiation were the expression levels of RELN, with almost 100-fold difference between the sexes, and MASH1, with more than 1,000-fold increase in male cells. To evaluate whether some of the observed differences may be sex related, we measured the expression of gametologous genes located on the X- and Y-chromosome. Most noticeable was the increase of Y-encoded demethylases KDM6C (UTY) and KDM5D during differentiation of male cells. Our results indicate that attention should be paid to sex when planning neurogenesis and transplantation experiments.