CXCL12/SDF-1 dynamically regulates hematopoietic stem cell (HSC) attraction in the bone marrow (BM). Circadian regulation of bone formation and HSC traffic is relayed in bone and BM by β-adrenergic receptors (β-AR) expressed on HSCs, osteoblasts, and mesenchymal stem/progenitor cells. Circadian HSC release from the BM follows rhythmic secretion of norepinephrine from nerve terminals, β3-AR activation, and Cxcl12 downregulation, possibly from reduced Sp1 nuclear content. Here, we show that β-AR stimulation in stromal cells causes Sp1 degradation, partially mediated by the 26S proteasome. Inverted trends of circulating hematopoietic progenitors and BM Cxcl12 mRNA levels change acutely after light onset, shown to induce sympathetic efferent activity. In BM stromal cells, activation of β3-AR downregulates Cxcl12, whereas β2-AR stimulation induces clock gene expression. Double deficiency in β2- and β3-ARs compromises enforced mobilization. Therefore, β2- and β3-ARs have specific roles in stromal cells and cooperate during progenitor mobilization.