Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis. These cells are classically defined as a subset of morphologically defined A single (A_s) spermatogonia, which can produce more SSCs or they can give rise to nonstem A_s cells that, upon replication, generate A paired (A_pr) and then A aligned (A_al) spermatogonia. These latter two cell types, along with the nonstem A_s cells, function as transit-amplifying progenitor cells. It is known that glial cell line-derived neurotrophic factor (GDNF) is essential for maintaining all of these cells, but it is unknown if or how the responses of these cells change as they progress down the pathway to differentiated type A1 spermatogonia. We address this issue by using a chemical-genetic approach to inhibit GDNF signaling in vivo and an in vitro approach to increase GDNF stimulation. We show that inhibition for 2 days suppresses replication of A_s, A_pr, and A_al spermatogonia to an equal extent, whereas stimulation by GDNF preferentially increases replication of A_s and A_pr spermatogonia. We also test if inhibiting GDNF signaling causes A_s, A_pr, and A_al spermatogonia to express Kit, an essential step in their differentiation into type A1 spermatogonia. Inhibition for 3 or 7 days produces a progressive increase in the percentages of A_s, A_pr, and A_al undergoing differentiation, with the largest increase observed in A_al spermatogonia. Finally, we demonstrate that numbers of SSCs decrease more slowly than numbers of progenitor spermatogonia when GDNF signaling is inhibited. Taken together, these data suggest that there are significant changes in the responses to GDNF as SSCs give rise to progenitor spermatogonia, which replicate and gradually differentiate into type A1 spermatogonia.