Limited cell numbers in a unit restricts cord blood transplantation (CBT) in adults. We evaluated whether cotransplantation of placental mesenchymal stromal cells (MSCs) would enhance engraftment. Plastic adherent cells from placenta demonstrated typical characteristics of MSCs. In six individual experiments, 4 cohorts of 24 nonobese diabetic/severe combined immune deficient (NOD/SCID) mice were evaluated. Cohort 1 received 5 x 10(4) CD34+ cells from unit (U) one (SCBT); cohort 2 received 5 x 10(4) CD34+ cells from U1 + 4 x 10(4) MSCs (SCBT+MSCs); cohort 3 received 2.5 x 10(4) CD34+ cells from U1 + 2.5 x 10(4) CD34+ cells from U2 (double cord blood transplant [DCBT]); cohort 4 received 2.5 x 10(4) CD34+ cells from U1 + 2.5 x 10(4) CD34+ cells from U2 + 4 x 10(4) MSCs (DCBT+MSCs). Hematopoietic engraftment evaluated after 6 to 8 weeks, was similar in recipients of SCBT and DCBT. MSC cotransplantation demonstrated enhanced engraftment in DCBT (51.8 +/- 6.8% versus 14.9 +/- 6.5%; p = .04) with an increased trend in SCBT (48.7 +/- 7.7% versus 17.5 +/- 6.1%; p = .07). In DCBT, cotransplantation of placental MSCs reduced single cord dominance. Self-renewal capacity was assessed by serial transplantation in secondary recipients infused with engrafted human cells from primary mice transplanted with or without MSCs. In secondary transplant experiments, 13 of 17 evaluable mice engrafted at levels of 1% to 6.5%. Despite enhanced engraftment in primary mice, long-term engraftment capacity was unaltered with MSC cotransplantation. Imaging studies showed MSCs migrated to pelvic region and improved cord blood (CB) CD34+ homing. Cotransplantation of placental MSCs enhanced cord blood engraftment and may act by improving homing of CD34+ cells.