Glioblastoma (GBM) represents an extremely aggressive brain malignancy with limited treatment options, difficult prognosis and a highly heterogeneous cellular architecture, including a subpopulation of cancer stem-like cells (CSCs). These CSCs frequently rely on developmental signaling pathways such as Sonic Hedgehog (SHH), which are typically dormant in adult tissue but reactivated in tumors. This study aimed to investigate how SHH pathway inhibition affects both bulk GBM cells (GBMCs) and CD133 + GBM cells (GBM CSCs), with particular emphasis on the influence of astrocyte co-culture, which more closely mimics the brain tumor microenvironment. GBMCs and GBM CSCs were cultured in mono- and astrocyte co-culture systems. They were evaluated through RT-qPCR, immunofluorescence staining, ELISA, TUNEL assay, and cell cycle analysis. By comparing treatment and culture context independently, cyclopamine-mediated SHH inhibition and astrocyte-depending signals use distinct but interacting effects on cell behavior. Cyclopamine treatment altered SHH pathway activity in a context-dependent manner, while astrocyte co-culture independently modulated GLI1, GLI3, and SUFU expression. GBM CSCs exhibited higher SHH secretion in monoculture, which was attenuated under co-culture with cyclopamine. Cell cycle analysis revealed G2/M arrest in GBMCs and G0/G1 arrest in CSCs, with astrocyte co-culture shifting CSCs toward G2/M. Apoptotic gene expression and TUNEL staining indicated enhanced extrinsic apoptosis (via CASP8) in CSCs, further intensified by SHH inhibition and co-culture. Astrocyte co-culture significantly modulates the molecular and phenotypic response of GBM cells to SHH inhibition, reshaping apoptotic and proliferative behaviors in both CSCs and bulk populations. These findings highlight the critical importance of the tumor microenvironment in therapeutic response and suggest that effective targeting of SHH signaling may require models that account for astroglial interactions.