Introduction: the adult hippocampal neurogenic niche relies on the activation of reversibly quiescent neural stem cells (NSCs). Thyroid hormones are critical regulators of neurogenesis, but their role in balancing NSC quiescence and activation has been little explored. Objective: this study examined whether thyroxine (L-T4) modulates this balance. Methodology: NSCs were isolated from 30-day-old C57BL/6J mice, cultured as neurospheres, and treated for 72 h with 1 nM or 5 nM L-T4 under two conditions: proliferation (FGF2, 20 ng/mL) or quiescence (FGF2 + BMP4, 30 ng/mL). The percentage of positive NSCs expressing Nestin, Ki67, and cyclin D1 was analysed. In proliferation conditions, both T4 concentrations increased Nestin+ cells without altering Cyclin D1, while 5 nM T4 reduced Ki67+ cells. In quiescence conditions, both doses decreased Nestin+ and Ki67+ cells, and 5 nM T4 increased Cyclin D1+ NSCs. Conclusion: These findings indicate that NSCs are sensitive to T4 levels in both proliferative and quiescent states. T4 promotes NSCs identity under proliferation conditions, but high T4 reduces proliferation, potentially preserving the NSCs pool. Under quiescence conditions, T4 may either prolong the cell cycle and reinforce quiescence or induce loss of stemness and differentiation. Altogether, our data suggest that thyroid hormones may be key modulators of the quiescence–activation balance, essential for sustaining hippocampal neurogenesis.