Cancer stem cells (CSCs) or circulating tumor cells play an important role in tumor initiation, invasion, metastasis and resistance to anticancer therapies. Therapies that target gastric tumor CSCs have potential clinical application for preventing malignant gastric tumor progression and metastasis. We isolated CD44+ gastric cancer cells from the gastric cancer cell line AGS and Hs746T cells and maintained the cells in a novel stem cell culture. The cells were kept in an undifferentiated proliferative state and we characterized their cancer stem cell properties and chemotherapy-resistance behavior. The CD44+ cancer cells were also co-cultured with human adipose stem cells (ADSCs) to determine the chemotherapy-promotion effects of the adipose cells on the CD44+ cancer cells. The CD44+ gastric cancer cell model is a non-adhesion, 3-dimensional, spheroid phenotype. The non-adherent CD44+ cells have cancer stem cell properties and are highly chemo-resistant. However, these cells regained chemo-sensitivity when re-attached to an extracellular matrix-coated attachment surface. The human adipose stem cells significantly promoted the chemo-sensitivity of the non-adherent CD44+ gastric cancer cells. Integrin α2/β2 and the Wnt signaling pathways are involved in the mechanisms. We concluded that the in vitro non-adherent CD44+ gastric cancer cell model resembles the circulating gastric tumor cells in vivo. Introduction of an appropriate attachment surface significantly promotes chemo-sensitivity of the non-adherent CD44+ gastric cancer cells. The human adipose stem cells function as a 'living vehicle surface' for such a purpose in vivo.